US20110077286A1 - Oligonucleotide duplexes comprising dna-like and rna-like nucleotides and uses thereof - Google Patents
Oligonucleotide duplexes comprising dna-like and rna-like nucleotides and uses thereof Download PDFInfo
- Publication number
- US20110077286A1 US20110077286A1 US12/996,362 US99636209A US2011077286A1 US 20110077286 A1 US20110077286 A1 US 20110077286A1 US 99636209 A US99636209 A US 99636209A US 2011077286 A1 US2011077286 A1 US 2011077286A1
- Authority
- US
- United States
- Prior art keywords
- rna
- ana
- sirna
- oligonucleotide pair
- nucleic acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 108091034117 Oligonucleotide Proteins 0.000 title claims abstract description 150
- 239000002773 nucleotide Substances 0.000 title claims abstract description 74
- 125000003729 nucleotide group Chemical group 0.000 title claims abstract description 73
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 102
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 102
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 100
- 230000014509 gene expression Effects 0.000 claims abstract description 31
- 125000002652 ribonucleotide group Chemical class 0.000 claims abstract description 8
- 108091028664 Ribonucleotide Proteins 0.000 claims abstract description 7
- 239000002214 arabinonucleotide Substances 0.000 claims abstract description 7
- 239000002336 ribonucleotide Substances 0.000 claims abstract description 7
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 214
- 230000000692 anti-sense effect Effects 0.000 claims description 127
- 108091081021 Sense strand Proteins 0.000 claims description 80
- 239000000203 mixture Substances 0.000 claims description 39
- 238000000034 method Methods 0.000 claims description 38
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 30
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 30
- 229920001184 polypeptide Polymers 0.000 claims description 29
- 108020004414 DNA Proteins 0.000 claims description 17
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 16
- 230000003247 decreasing effect Effects 0.000 claims description 15
- 201000010099 disease Diseases 0.000 claims description 15
- 230000000295 complement effect Effects 0.000 claims description 11
- 230000000593 degrading effect Effects 0.000 claims description 7
- 239000003937 drug carrier Substances 0.000 claims description 6
- 229910052736 halogen Inorganic materials 0.000 claims description 4
- 150000002367 halogens Chemical group 0.000 claims description 4
- 239000005547 deoxyribonucleotide Substances 0.000 claims description 3
- 125000002637 deoxyribonucleotide group Chemical group 0.000 claims description 3
- PXGOKWXKJXAPGV-UHFFFAOYSA-N Fluorine Chemical compound FF PXGOKWXKJXAPGV-UHFFFAOYSA-N 0.000 claims description 2
- 229910052731 fluorine Inorganic materials 0.000 claims description 2
- 239000011737 fluorine Substances 0.000 claims description 2
- 108020004459 Small interfering RNA Proteins 0.000 abstract description 159
- 239000004055 small Interfering RNA Substances 0.000 abstract description 117
- 108090000623 proteins and genes Proteins 0.000 abstract description 49
- 230000030279 gene silencing Effects 0.000 abstract description 24
- 238000005516 engineering process Methods 0.000 abstract description 4
- 210000004027 cell Anatomy 0.000 description 68
- 230000000694 effects Effects 0.000 description 26
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 description 23
- 230000009368 gene silencing by RNA Effects 0.000 description 23
- 230000036515 potency Effects 0.000 description 22
- 230000004048 modification Effects 0.000 description 21
- 238000012986 modification Methods 0.000 description 21
- 238000004519 manufacturing process Methods 0.000 description 20
- 239000003153 chemical reaction reagent Substances 0.000 description 19
- 230000003389 potentiating effect Effects 0.000 description 19
- 238000013461 design Methods 0.000 description 18
- 238000001890 transfection Methods 0.000 description 18
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 17
- 238000003197 gene knockdown Methods 0.000 description 16
- 235000000346 sugar Nutrition 0.000 description 15
- 238000011282 treatment Methods 0.000 description 15
- 108050000946 Eukaryotic translation initiation factor 4E-binding protein 1 Proteins 0.000 description 14
- 102100022466 Eukaryotic translation initiation factor 4E-binding protein 1 Human genes 0.000 description 14
- 239000003814 drug Substances 0.000 description 13
- -1 methylene(methylimino) Chemical class 0.000 description 13
- 238000012226 gene silencing method Methods 0.000 description 12
- 229910019142 PO4 Inorganic materials 0.000 description 11
- 230000015572 biosynthetic process Effects 0.000 description 11
- 150000001875 compounds Chemical class 0.000 description 11
- 239000002777 nucleoside Substances 0.000 description 11
- 238000002360 preparation method Methods 0.000 description 11
- 102000004169 proteins and genes Human genes 0.000 description 11
- 239000010452 phosphate Substances 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- 108020004999 messenger RNA Proteins 0.000 description 9
- 150000003833 nucleoside derivatives Chemical class 0.000 description 9
- 239000000047 product Substances 0.000 description 9
- 238000003786 synthesis reaction Methods 0.000 description 9
- 230000008685 targeting Effects 0.000 description 9
- 108090000331 Firefly luciferases Proteins 0.000 description 8
- 108060001084 Luciferase Proteins 0.000 description 8
- 239000005089 Luciferase Substances 0.000 description 8
- 102000000574 RNA-Induced Silencing Complex Human genes 0.000 description 8
- 108010016790 RNA-Induced Silencing Complex Proteins 0.000 description 8
- 239000003795 chemical substances by application Substances 0.000 description 8
- 229940115272 polyinosinic:polycytidylic acid Drugs 0.000 description 8
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 7
- 230000005764 inhibitory process Effects 0.000 description 7
- 150000003212 purines Chemical class 0.000 description 7
- 150000003230 pyrimidines Chemical class 0.000 description 7
- 239000013598 vector Substances 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 238000007385 chemical modification Methods 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 238000009472 formulation Methods 0.000 description 6
- 239000012634 fragment Substances 0.000 description 6
- 230000002401 inhibitory effect Effects 0.000 description 6
- 208000032839 leukemia Diseases 0.000 description 6
- 125000001921 locked nucleotide group Chemical group 0.000 description 6
- 238000002515 oligonucleotide synthesis Methods 0.000 description 6
- 210000001519 tissue Anatomy 0.000 description 6
- 101710082153 Eukaryotic translation initiation factor 4E-binding protein 2 Proteins 0.000 description 5
- 102100022447 Eukaryotic translation initiation factor 4E-binding protein 2 Human genes 0.000 description 5
- 241001529936 Murinae Species 0.000 description 5
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 5
- 239000002253 acid Substances 0.000 description 5
- 150000007513 acids Chemical class 0.000 description 5
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 5
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 5
- 238000009396 hybridization Methods 0.000 description 5
- 238000000338 in vitro Methods 0.000 description 5
- 210000001161 mammalian embryo Anatomy 0.000 description 5
- 230000001404 mediated effect Effects 0.000 description 5
- 108091027963 non-coding RNA Proteins 0.000 description 5
- 102000042567 non-coding RNA Human genes 0.000 description 5
- 230000003647 oxidation Effects 0.000 description 5
- 238000007254 oxidation reaction Methods 0.000 description 5
- 150000004713 phosphodiesters Chemical class 0.000 description 5
- 229920001223 polyethylene glycol Polymers 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- PEHVGBZKEYRQSX-UHFFFAOYSA-N 7-deaza-adenine Chemical compound NC1=NC=NC2=C1C=CN2 PEHVGBZKEYRQSX-UHFFFAOYSA-N 0.000 description 4
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 4
- 101000678286 Danio rerio Eukaryotic translation initiation factor 4E-binding protein 3-like Proteins 0.000 description 4
- 101000800913 Dictyostelium discoideum Eukaryotic translation initiation factor 4E-1A-binding protein homolog Proteins 0.000 description 4
- 101000800906 Drosophila melanogaster Eukaryotic translation initiation factor 4E-binding protein Proteins 0.000 description 4
- 239000002202 Polyethylene glycol Substances 0.000 description 4
- 108010087776 Proto-Oncogene Proteins c-myb Proteins 0.000 description 4
- 102000009096 Proto-Oncogene Proteins c-myb Human genes 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 4
- 125000000217 alkyl group Chemical group 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 239000006185 dispersion Substances 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 125000000623 heterocyclic group Chemical group 0.000 description 4
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 4
- 238000001727 in vivo Methods 0.000 description 4
- DRAVOWXCEBXPTN-UHFFFAOYSA-N isoguanine Chemical compound NC1=NC(=O)NC2=C1NC=N2 DRAVOWXCEBXPTN-UHFFFAOYSA-N 0.000 description 4
- 238000011068 loading method Methods 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 210000000056 organ Anatomy 0.000 description 4
- 230000002018 overexpression Effects 0.000 description 4
- 125000006239 protecting group Chemical group 0.000 description 4
- 230000009467 reduction Effects 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 230000001225 therapeutic effect Effects 0.000 description 4
- RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical compound [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 0.000 description 4
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 4
- WYWHKKSPHMUBEB-UHFFFAOYSA-N tioguanine Chemical compound N1C(N)=NC(=S)C2=C1N=CN2 WYWHKKSPHMUBEB-UHFFFAOYSA-N 0.000 description 4
- GONFBOIJNUKKST-UHFFFAOYSA-N 5-ethylsulfanyl-2h-tetrazole Chemical compound CCSC=1N=NNN=1 GONFBOIJNUKKST-UHFFFAOYSA-N 0.000 description 3
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 3
- 102100029172 Choline-phosphate cytidylyltransferase A Human genes 0.000 description 3
- 101710100763 Choline-phosphate cytidylyltransferase A Proteins 0.000 description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 239000004952 Polyamide Substances 0.000 description 3
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 3
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 3
- 108091027967 Small hairpin RNA Proteins 0.000 description 3
- RYYWUUFWQRZTIU-UHFFFAOYSA-N Thiophosphoric acid Chemical class OP(O)(S)=O RYYWUUFWQRZTIU-UHFFFAOYSA-N 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 125000003275 alpha amino acid group Chemical group 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 239000000074 antisense oligonucleotide Substances 0.000 description 3
- 238000012230 antisense oligonucleotides Methods 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 238000001142 circular dichroism spectrum Methods 0.000 description 3
- 230000008878 coupling Effects 0.000 description 3
- 238000010168 coupling process Methods 0.000 description 3
- 238000005859 coupling reaction Methods 0.000 description 3
- 230000001186 cumulative effect Effects 0.000 description 3
- 229940104302 cytosine Drugs 0.000 description 3
- 239000002612 dispersion medium Substances 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 239000004615 ingredient Substances 0.000 description 3
- 239000002502 liposome Substances 0.000 description 3
- 239000000546 pharmaceutical excipient Substances 0.000 description 3
- 235000021317 phosphate Nutrition 0.000 description 3
- 229920002647 polyamide Polymers 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 230000002265 prevention Effects 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- UHUHBFMZVCOEOV-UHFFFAOYSA-N 1h-imidazo[4,5-c]pyridin-4-amine Chemical compound NC1=NC=CC2=C1N=CN2 UHUHBFMZVCOEOV-UHFFFAOYSA-N 0.000 description 2
- XGDRLCRGKUCBQL-UHFFFAOYSA-N 1h-imidazole-4,5-dicarbonitrile Chemical compound N#CC=1N=CNC=1C#N XGDRLCRGKUCBQL-UHFFFAOYSA-N 0.000 description 2
- ZLAQATDNGLKIEV-UHFFFAOYSA-N 5-methyl-2-sulfanylidene-1h-pyrimidin-4-one Chemical compound CC1=CNC(=S)NC1=O ZLAQATDNGLKIEV-UHFFFAOYSA-N 0.000 description 2
- GSPMCUUYNASDHM-UHFFFAOYSA-N 5-methyl-4-sulfanylidene-1h-pyrimidin-2-one Chemical compound CC1=CNC(=O)N=C1S GSPMCUUYNASDHM-UHFFFAOYSA-N 0.000 description 2
- LRSASMSXMSNRBT-UHFFFAOYSA-N 5-methylcytosine Chemical compound CC1=CNC(=O)N=C1N LRSASMSXMSNRBT-UHFFFAOYSA-N 0.000 description 2
- LOSIULRWFAEMFL-UHFFFAOYSA-N 7-deazaguanine Chemical compound O=C1NC(N)=NC2=C1CC=N2 LOSIULRWFAEMFL-UHFFFAOYSA-N 0.000 description 2
- IBCFXJUTCBHRBV-UHFFFAOYSA-N 9-deazaadenine Chemical compound NC1=NC=NC2=CC=N[C]12 IBCFXJUTCBHRBV-UHFFFAOYSA-N 0.000 description 2
- GTJHDGVPBSHFSU-UHFFFAOYSA-N 9-deazaguanine Chemical compound O=C1NC(N)=NC2=CC=N[C]21 GTJHDGVPBSHFSU-UHFFFAOYSA-N 0.000 description 2
- MSSXOMSJDRHRMC-UHFFFAOYSA-N 9H-purine-2,6-diamine Chemical compound NC1=NC(N)=C2NC=NC2=N1 MSSXOMSJDRHRMC-UHFFFAOYSA-N 0.000 description 2
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 2
- 229930024421 Adenine Natural products 0.000 description 2
- 108091026890 Coding region Proteins 0.000 description 2
- 229920000742 Cotton Polymers 0.000 description 2
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 description 2
- UGQMRVRMYYASKQ-KQYNXXCUSA-N Inosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC(O)=C2N=C1 UGQMRVRMYYASKQ-KQYNXXCUSA-N 0.000 description 2
- 229930010555 Inosine Natural products 0.000 description 2
- 108010050904 Interferons Proteins 0.000 description 2
- 102000014150 Interferons Human genes 0.000 description 2
- 241000254158 Lampyridae Species 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 108020004711 Nucleic Acid Probes Proteins 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- 102000003661 Ribonuclease III Human genes 0.000 description 2
- 108010057163 Ribonuclease III Proteins 0.000 description 2
- 102000039471 Small Nuclear RNA Human genes 0.000 description 2
- 108020004566 Transfer RNA Proteins 0.000 description 2
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 229960000643 adenine Drugs 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 230000004663 cell proliferation Effects 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 125000003636 chemical group Chemical group 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 230000002939 deleterious effect Effects 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000001627 detrimental effect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 210000003754 fetus Anatomy 0.000 description 2
- 230000037440 gene silencing effect Effects 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- 230000003308 immunostimulating effect Effects 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 229960003786 inosine Drugs 0.000 description 2
- 229940079322 interferon Drugs 0.000 description 2
- 230000001788 irregular Effects 0.000 description 2
- 239000007951 isotonicity adjuster Substances 0.000 description 2
- 239000003446 ligand Substances 0.000 description 2
- 125000005647 linker group Chemical group 0.000 description 2
- 238000004949 mass spectrometry Methods 0.000 description 2
- 238000002844 melting Methods 0.000 description 2
- 230000008018 melting Effects 0.000 description 2
- GLVAUDGFNGKCSF-UHFFFAOYSA-N mercaptopurine Chemical compound S=C1NC=NC2=C1NC=N2 GLVAUDGFNGKCSF-UHFFFAOYSA-N 0.000 description 2
- 239000000178 monomer Substances 0.000 description 2
- 239000002853 nucleic acid probe Substances 0.000 description 2
- 210000003463 organelle Anatomy 0.000 description 2
- 239000008194 pharmaceutical composition Substances 0.000 description 2
- 238000006366 phosphorylation reaction Methods 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 108020004418 ribosomal RNA Proteins 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 238000003252 siRNA assay Methods 0.000 description 2
- 108091029842 small nuclear ribonucleic acid Proteins 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000007790 solid phase Substances 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 229940113082 thymine Drugs 0.000 description 2
- 229960003087 tioguanine Drugs 0.000 description 2
- 229940035893 uracil Drugs 0.000 description 2
- 239000003981 vehicle Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 description 1
- JVSFQJZRHXAUGT-UHFFFAOYSA-N 2,2-dimethylpropanoyl chloride Chemical compound CC(C)(C)C(Cl)=O JVSFQJZRHXAUGT-UHFFFAOYSA-N 0.000 description 1
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- 108091092742 A-DNA Proteins 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
- 241000589158 Agrobacterium Species 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 108020000948 Antisense Oligonucleotides Proteins 0.000 description 1
- 102100037435 Antiviral innate immune response receptor RIG-I Human genes 0.000 description 1
- 101710127675 Antiviral innate immune response receptor RIG-I Proteins 0.000 description 1
- 108010039627 Aprotinin Proteins 0.000 description 1
- KPINOLRMYARTNG-FWYCZPIQSA-N COC[C@H]1O[C@@H](C)[C@@H](F)C1OC Chemical compound COC[C@H]1O[C@@H](C)[C@@H](F)C1OC KPINOLRMYARTNG-FWYCZPIQSA-N 0.000 description 1
- UMNPKGLZSHYVMY-YRDQVMKSSA-N COC[C@H]1O[C@@H](C)[C@@H](F)C1OC.COC[C@H]1O[C@@H](C)[C@@H](O)C1OC.COC[C@]12CO[C@@H](C1OC)[C@H](C)O2 Chemical compound COC[C@H]1O[C@@H](C)[C@@H](F)C1OC.COC[C@H]1O[C@@H](C)[C@@H](O)C1OC.COC[C@]12CO[C@@H](C1OC)[C@H](C)O2 UMNPKGLZSHYVMY-YRDQVMKSSA-N 0.000 description 1
- QCMYYKRYFNMIEC-UHFFFAOYSA-N COP(O)=O Chemical class COP(O)=O QCMYYKRYFNMIEC-UHFFFAOYSA-N 0.000 description 1
- 101100457838 Caenorhabditis elegans mod-1 gene Proteins 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 108010001857 Cell Surface Receptors Proteins 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 108700039887 Essential Genes Proteins 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 108091027305 Heteroduplex Proteins 0.000 description 1
- 101000678280 Homo sapiens Eukaryotic translation initiation factor 4E-binding protein 1 Proteins 0.000 description 1
- 101000678283 Homo sapiens Eukaryotic translation initiation factor 4E-binding protein 2 Proteins 0.000 description 1
- 101001082073 Homo sapiens Interferon-induced helicase C domain-containing protein 1 Proteins 0.000 description 1
- 101000582320 Homo sapiens Neurogenic differentiation factor 6 Proteins 0.000 description 1
- 108010047761 Interferon-alpha Proteins 0.000 description 1
- 102000006992 Interferon-alpha Human genes 0.000 description 1
- 102100027353 Interferon-induced helicase C domain-containing protein 1 Human genes 0.000 description 1
- 239000005909 Kieselgur Substances 0.000 description 1
- GDBQQVLCIARPGH-UHFFFAOYSA-N Leupeptin Natural products CC(C)CC(NC(C)=O)C(=O)NC(CC(C)C)C(=O)NC(C=O)CCCN=C(N)N GDBQQVLCIARPGH-UHFFFAOYSA-N 0.000 description 1
- 101150110972 ME1 gene Proteins 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 102100030589 Neurogenic differentiation factor 6 Human genes 0.000 description 1
- 102100032139 Neuroguidin Human genes 0.000 description 1
- 101710203741 Neuroguidin Proteins 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 108091005461 Nucleic proteins Chemical group 0.000 description 1
- 229910004679 ONO2 Inorganic materials 0.000 description 1
- 108091093037 Peptide nucleic acid Proteins 0.000 description 1
- 229920002732 Polyanhydride Polymers 0.000 description 1
- 229920000954 Polyglycolide Polymers 0.000 description 1
- 229920001710 Polyorthoester Polymers 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 102000052575 Proto-Oncogene Human genes 0.000 description 1
- 108700020978 Proto-Oncogene Proteins 0.000 description 1
- 108010052090 Renilla Luciferases Proteins 0.000 description 1
- 108700008625 Reporter Genes Proteins 0.000 description 1
- 108091027981 Response element Proteins 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 1
- 101800002899 Soluble alkaline phosphatase Proteins 0.000 description 1
- 101710137500 T7 RNA polymerase Proteins 0.000 description 1
- 108700019146 Transgenes Proteins 0.000 description 1
- LZAPBCCIWLYTHI-UHFFFAOYSA-N [2-(1,6-dicyano-3-methylhexan-3-yl)-2-propan-2-ylhydrazinyl]phosphonous acid Chemical compound CC(C)N(C(C)(CCCC#N)CCC#N)NP(O)O LZAPBCCIWLYTHI-UHFFFAOYSA-N 0.000 description 1
- HMNZFMSWFCAGGW-XPWSMXQVSA-N [3-[hydroxy(2-hydroxyethoxy)phosphoryl]oxy-2-[(e)-octadec-9-enoyl]oxypropyl] (e)-octadec-9-enoate Chemical compound CCCCCCCC\C=C\CCCCCCCC(=O)OCC(COP(O)(=O)OCCO)OC(=O)CCCCCCC\C=C\CCCCCCCC HMNZFMSWFCAGGW-XPWSMXQVSA-N 0.000 description 1
- 239000003070 absorption delaying agent Substances 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 1
- 229940127024 acid based drug Drugs 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 125000002015 acyclic group Chemical group 0.000 description 1
- 210000001789 adipocyte Anatomy 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 125000002877 alkyl aryl group Chemical group 0.000 description 1
- 150000001413 amino acids Chemical group 0.000 description 1
- 125000005122 aminoalkylamino group Chemical group 0.000 description 1
- 239000000908 ammonium hydroxide Substances 0.000 description 1
- 238000005571 anion exchange chromatography Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 229940121375 antifungal agent Drugs 0.000 description 1
- 239000003429 antifungal agent Substances 0.000 description 1
- 229960004405 aprotinin Drugs 0.000 description 1
- 125000003710 aryl alkyl group Chemical group 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 210000003651 basophil Anatomy 0.000 description 1
- 210000000227 basophil cell of anterior lobe of hypophysis Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229920000249 biocompatible polymer Polymers 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000029918 bioluminescence Effects 0.000 description 1
- 238000005415 bioluminescence Methods 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- DQXBYHZEEUGOBF-UHFFFAOYSA-N but-3-enoic acid;ethene Chemical compound C=C.OC(=O)CC=C DQXBYHZEEUGOBF-UHFFFAOYSA-N 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 125000001369 canonical nucleoside group Chemical group 0.000 description 1
- MPBRYMWMMKKRGC-UHFFFAOYSA-M carbocyanin DBTC Chemical compound [Br-].C1=CC=CC2=C([N+](=C(C=C(C)C=C3N(C4=C5C=CC=CC5=CC=C4S3)CC)S3)CC)C3=CC=C21 MPBRYMWMMKKRGC-UHFFFAOYSA-M 0.000 description 1
- 210000004413 cardiac myocyte Anatomy 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 230000004700 cellular uptake Effects 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 210000001612 chondrocyte Anatomy 0.000 description 1
- 238000002983 circular dichroism Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 239000005289 controlled pore glass Substances 0.000 description 1
- 238000013270 controlled release Methods 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 125000000753 cycloalkyl group Chemical group 0.000 description 1
- 125000001995 cyclobutyl group Chemical group [H]C1([H])C([H])([H])C([H])(*)C1([H])[H] 0.000 description 1
- 230000001934 delay Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000010511 deprotection reaction Methods 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- 239000013024 dilution buffer Substances 0.000 description 1
- XPPKVPWEQAFLFU-UHFFFAOYSA-J diphosphate(4-) Chemical compound [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 description 1
- 235000011180 diphosphates Nutrition 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 238000009510 drug design Methods 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 230000002124 endocrine Effects 0.000 description 1
- 210000003372 endocrine gland Anatomy 0.000 description 1
- 210000003038 endothelium Anatomy 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 210000003979 eosinophil Anatomy 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 239000005038 ethylene vinyl acetate Substances 0.000 description 1
- 210000003499 exocrine gland Anatomy 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 230000005714 functional activity Effects 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 210000004602 germ cell Anatomy 0.000 description 1
- 210000003714 granulocyte Anatomy 0.000 description 1
- 238000009499 grossing Methods 0.000 description 1
- 210000003494 hepatocyte Anatomy 0.000 description 1
- 125000000592 heterocycloalkyl group Chemical group 0.000 description 1
- 210000003630 histaminocyte Anatomy 0.000 description 1
- 102000043464 human EIF4EBP1 Human genes 0.000 description 1
- 102000053005 human EIF4EBP2 Human genes 0.000 description 1
- 238000010185 immunofluorescence analysis Methods 0.000 description 1
- 239000007943 implant Substances 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- ZPNFWUPYTFPOJU-LPYSRVMUSA-N iniprol Chemical compound C([C@H]1C(=O)NCC(=O)NCC(=O)N[C@H]2CSSC[C@H]3C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(N[C@H](C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC=4C=CC=CC=4)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC=4C=CC=CC=4)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC2=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC=2C=CC=CC=2)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H]2N(CCC2)C(=O)[C@@H](N)CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N2[C@@H](CCC2)C(=O)N2[C@@H](CCC2)C(=O)N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N2[C@@H](CCC2)C(=O)N3)C(=O)NCC(=O)NCC(=O)N[C@@H](C)C(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@H](C(=O)N1)C(C)C)[C@@H](C)O)[C@@H](C)CC)=O)[C@@H](C)CC)C1=CC=C(O)C=C1 ZPNFWUPYTFPOJU-LPYSRVMUSA-N 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 239000007972 injectable composition Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 239000000138 intercalating agent Substances 0.000 description 1
- 230000010468 interferon response Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 239000002608 ionic liquid Substances 0.000 description 1
- 210000002510 keratinocyte Anatomy 0.000 description 1
- 238000011813 knockout mouse model Methods 0.000 description 1
- 238000011005 laboratory method Methods 0.000 description 1
- 239000000787 lecithin Substances 0.000 description 1
- 229940067606 lecithin Drugs 0.000 description 1
- 235000010445 lecithin Nutrition 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- GDBQQVLCIARPGH-ULQDDVLXSA-N leupeptin Chemical compound CC(C)C[C@H](NC(C)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C=O)CCCN=C(N)N GDBQQVLCIARPGH-ULQDDVLXSA-N 0.000 description 1
- 108010052968 leupeptin Proteins 0.000 description 1
- 125000005524 levulinyl group Chemical group 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- MHCFAGZWMAWTNR-UHFFFAOYSA-M lithium perchlorate Chemical compound [Li+].[O-]Cl(=O)(=O)=O MHCFAGZWMAWTNR-UHFFFAOYSA-M 0.000 description 1
- 229910001486 lithium perchlorate Inorganic materials 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 210000003593 megakaryocyte Anatomy 0.000 description 1
- 102000006240 membrane receptors Human genes 0.000 description 1
- 125000001570 methylene group Chemical group [H]C([H])([*:1])[*:2] 0.000 description 1
- YACKEPLHDIMKIO-UHFFFAOYSA-N methylphosphonic acid Chemical compound CP(O)(O)=O YACKEPLHDIMKIO-UHFFFAOYSA-N 0.000 description 1
- 239000004530 micro-emulsion Substances 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 125000004573 morpholin-4-yl group Chemical group N1(CCOCC1)* 0.000 description 1
- 210000000107 myocyte Anatomy 0.000 description 1
- OHDXDNUPVVYWOV-UHFFFAOYSA-N n-methyl-1-(2-naphthalen-1-ylsulfanylphenyl)methanamine Chemical compound CNCC1=CC=CC=C1SC1=CC=CC2=CC=CC=C12 OHDXDNUPVVYWOV-UHFFFAOYSA-N 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 210000004498 neuroglial cell Anatomy 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 125000001893 nitrooxy group Chemical group [O-][N+](=O)O* 0.000 description 1
- 230000000269 nucleophilic effect Effects 0.000 description 1
- 230000009437 off-target effect Effects 0.000 description 1
- 125000001181 organosilyl group Chemical group [SiH3]* 0.000 description 1
- 210000000963 osteoblast Anatomy 0.000 description 1
- 210000002997 osteoclast Anatomy 0.000 description 1
- 239000007800 oxidant agent Substances 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 229950000964 pepstatin Drugs 0.000 description 1
- 108010091212 pepstatin Proteins 0.000 description 1
- FAXGPCHRFPCXOO-LXTPJMTPSA-N pepstatin A Chemical compound OC(=O)C[C@H](O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)C[C@H](O)[C@H](CC(C)C)NC(=O)[C@H](C(C)C)NC(=O)[C@H](C(C)C)NC(=O)CC(C)C FAXGPCHRFPCXOO-LXTPJMTPSA-N 0.000 description 1
- 230000003285 pharmacodynamic effect Effects 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
- 150000008300 phosphoramidites Chemical class 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 229920001200 poly(ethylene-vinyl acetate) Polymers 0.000 description 1
- 229920000747 poly(lactic acid) Polymers 0.000 description 1
- 239000004633 polyglycolic acid Substances 0.000 description 1
- 239000004626 polylactic acid Substances 0.000 description 1
- 229920005862 polyol Polymers 0.000 description 1
- 150000003077 polyols Chemical class 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000001124 posttranscriptional effect Effects 0.000 description 1
- 230000032361 posttranscriptional gene silencing Effects 0.000 description 1
- SCVFZCLFOSHCOH-UHFFFAOYSA-M potassium acetate Chemical compound [K+].CC([O-])=O SCVFZCLFOSHCOH-UHFFFAOYSA-M 0.000 description 1
- GUUBJKMBDULZTE-UHFFFAOYSA-M potassium;2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid;hydroxide Chemical compound [OH-].[K+].OCCN1CCN(CCS(O)(=O)=O)CC1 GUUBJKMBDULZTE-UHFFFAOYSA-M 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 238000002731 protein assay Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 125000006853 reporter group Chemical group 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 239000010703 silicon Substances 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- HBMJWWWQQXIZIP-UHFFFAOYSA-N silicon carbide Chemical compound [Si+]#[C-] HBMJWWWQQXIZIP-UHFFFAOYSA-N 0.000 description 1
- 229910010271 silicon carbide Inorganic materials 0.000 description 1
- 125000003808 silyl group Chemical group [H][Si]([H])([H])[*] 0.000 description 1
- 230000000392 somatic effect Effects 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 150000005846 sugar alcohols Polymers 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 125000004434 sulfur atom Chemical group 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 239000011885 synergistic combination Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 150000003536 tetrazoles Chemical class 0.000 description 1
- 238000011287 therapeutic dose Methods 0.000 description 1
- 230000004797 therapeutic response Effects 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 125000000876 trifluoromethoxy group Chemical group FC(F)(F)O* 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 229910000404 tripotassium phosphate Inorganic materials 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- 125000002221 trityl group Chemical group [H]C1=C([H])C([H])=C([H])C([H])=C1C([*])(C1=C(C(=C(C(=C1[H])[H])[H])[H])[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 1
- 230000014567 type I interferon production Effects 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 238000009777 vacuum freeze-drying Methods 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/111—General methods applicable to biologically active non-coding nucleic acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering N.A.
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/32—Chemical structure of the sugar
- C12N2310/322—2'-R Modification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/32—Chemical structure of the sugar
- C12N2310/323—Chemical structure of the sugar modified ring structure
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2320/00—Applications; Uses
- C12N2320/30—Special therapeutic applications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2320/00—Applications; Uses
- C12N2320/50—Methods for regulating/modulating their activity
- C12N2320/51—Methods for regulating/modulating their activity modulating the chemical stability, e.g. nuclease-resistance
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2320/00—Applications; Uses
- C12N2320/50—Methods for regulating/modulating their activity
- C12N2320/53—Methods for regulating/modulating their activity reducing unwanted side-effects
Definitions
- the invention relates to oligonucleotides, methods for their preparation and uses thereof, such as for decreasing the level of a target nucleic acid in a cell, and/or silencing the expression of a nucleic acid or gene of interest using small interfering RNA (siRNA) technologies.
- siRNA small interfering RNA
- Gene silencing i.e., selectively blocking the expression of a gene of interest, may be effected via the introduction of an antisense oligonucleotide (AON) or small interfering RNA (siRNA) into an organism (Uhlmann, E. and Peyman, A. Chem. Rev. 1990, 90: 543-84; Braasch, D. A. and Corey, D. R. Biochemistry 2002, 41: 4503-4510; Opalinska, J. B. and Gewirtz, A. M. Nat. Rev. Drug Discov. 2002, 1: 503-14; Dorsett, Y. and Tuschl, T. Nat. Rev. Drug Discov. 2004, 3: 318-329).
- AON antisense oligonucleotide
- siRNA small interfering RNA
- siRNAs have poor serum stability, poor cellular uptake, and can elicit off-target and immunostimulatory side effects.
- Efforts to remedy these shortcomings have focused on the development of delivery vehicles for siRNAs, and on the development of chemically modified oligonucleotides with improved drug profiles.
- siRNA duplexes with extensive 2′F-ANA modification were found to have a significantly longer serum half-life than unmodified siRNAs.
- Modified siRNA duplexes containing 2′-fluoro-4′-thioarabinonucleotide (4′S-FANA) units were able to enter the RNAi pathway (Watts, J. K. et al. Nucl. Acids Res. 2007, 35: 1441-1451). One or two inserts internally in either strand gave duplexes of potency comparable to that of the control.
- the 4′S-FANA modification was also able to work with good efficiency in a duplex with a modified 2′F-ANA-RNA sense strand, demonstrating that 2′F-ANA (with its preference for southern and eastern conformations) can achieve synergy with 4′S-2′F-ANA (with its preference for northern conformations), in RNAi gene silencing.
- 2′F-RNA is another siRNA modification, and partial 2′F-RNA modification is tolerated throughout both the sense and antisense strands, and some fully-modified 2′F-RNA siRNAs are also active.
- 2′F-RNA-modified siRNA duplexes have significantly increased serum stability (Layzer, J. M. et al. RNA, 2004, 10: 766-771). 2′F-RNA also increases the binding affinity of the duplex.
- the invention relates to oligonucleotides, methods for their preparation and uses thereof, such as for decreasing the level of a target nucleic acid in a cell, and/or silencing the expression of a nucleic acid or gene of interest using small interfering RNA (siRNA) technologies.
- siRNA small interfering RNA
- the present invention provides an oligonucleotide pair which can form a duplex, comprising:
- the present invention provides an oligonucleotide pair which can form a duplex, comprising a sense strand and an antisense strand complementary to the sense strand, wherein the oligonucleotide pair comprises: (a) one or more 2′-substituted arabinonucleotides (ANA); and (b) (i) one or more 2′-substituted ribonucleotides (RNA), (ii) one or more locked nucleic acid nucleotides (LNA), or (iii) a combination of (i) and (ii).
- ANA 2′-substituted arabinonucleotides
- RNA 2′-substituted ribonucleotides
- LNA locked nucleic acid nucleotides
- the above-mentioned oligonucleotide pair comprises one or more 2′-substituted ANA and one or more 2′-substituted RNA. In another embodiment, the above-mentioned oligonucleotide pair comprises one or more 2′-substituted ANA and one or more LNA. In another embodiment, the above-mentioned oligonucleotide pair comprises one or more 2′-substituted ANA, one or more 2′-substituted RNA and one or more LNA.
- the above-mentioned 2′-substitutent is an halogen.
- the above-mentioned halogen is fluorine (F).
- the above-mentioned sense strand comprises: (i) 2′F-ANA only; (ii) 2′F-RNA only; (iii) a combination of 2′F-RNA and 2′F-ANA; (iv) RNA only; (v) a combination of 2′F-ANA and RNA; (vi) a combination of 2′F-ANA, RNA and LNA; or (vii) a combination of 2′F -ANA, 2′F-RNA and RNA.
- the above-mentioned antisense strand comprises: (i) 2′F-RNA only; (ii) RNA only; (iii) 2′F-ANA only; (iv) a combination of 2′F-RNA and 2′F-ANA; (v) a combination of 2′F-ANA and RNA; (vi) a combination of 2′F-ANA, RNA and LNA; or (vii) a combination of 2′F-ANA, 2′F-RNA and RNA.
- the above-mentioned sense strand and antisense strand have a length of 19 to 23 residues. In a further embodiment, the above-mentioned sense strand and antisense strand have a length of 21 residues.
- the above-mentioned sense strand, antisense strand, or both comprises an overhang at the 3′ end.
- the above-mentioned overhang is from 1 to 5 residues, in a further embodiment 2 residues.
- the above-mentioned overhang comprises deoxyribonucleotides (DNA), 2′F-ANA, or a combination thereof.
- the above-mentioned sense strand, antisense strand, or both is/are phosphorylated at the 5′ end.
- the above-mentioned antisense strand is phosphorylated at the 5′ end.
- the present invention provides a double-stranded siRNA-like molecule comprising the above-mentioned oligonucleotide pair.
- the above-mentioned sense and antisense strands are within an oligonucleotide of 15 to 80 nucleotides in length and such that the oligonucleotide or a portion thereof is capable of adopting an siRNA-like hairpin structure in which the sense and antisense strands form the stem of the hairpin structure.
- the present invention provides a composition comprising the above-mentioned oligonucleotide pair or the above-mentioned double-stranded siRNA-like molecule, and a pharmaceutically acceptable carrier.
- the present invention provides the above-mentioned oligonucleotide pair, the above-mentioned double-stranded siRNA-like molecule, or the above-mentioned composition, for decreasing the level of a target nucleic acid, or of a polypeptide encoded by said target nucleic acid, in a cell, wherein the sense strand of the oligonucleotide pair comprises a nucleobase sequence substantially identical to a nucleobase sequence of the target nucleic acid.
- the present invention provides the above-mentioned oligonucleotide pair, the above-mentioned double-stranded siRNA-like molecule, or the above-mentioned composition, for preventing or treating a disease or condition associated with the expression of a target nucleic acid, or of a polypeptide encoded by said target nucleic acid, in a subject, wherein the sense strand of the oligonucleotide pair comprises a nucleobase sequence substantially identical to a nucleobase sequence of the target nucleic acid.
- the present invention provides a method of degrading or decreasing the level of a target nucleic acid, or of decreasing the production or the level of a polypeptide encoded by said target nucleic acid, in a cell, the method comprising contacting the cell with the above-mentioned oligonucleotide pair, the above-mentioned double-stranded siRNA-like molecule, or the above-mentioned composition, wherein the sense strand of the oligonucleotide pair comprises a nucleobase sequence substantially identical to a nucleobase sequence of the target nucleic acid.
- the present invention provides a method of preventing or treating a disease or condition associated with the expression of a target nucleic acid, or of a polypeptide encoded by said target nucleic acid, in a subject, the method comprising administering to the subject an effective amount of the above-mentioned oligonucleotide pair, the above-mentioned double-stranded siRNA-like molecule, or the above-mentioned composition, wherein the sense strand of the oligonucleotide pair comprises a nucleobase sequence substantially identical to a nucleobase sequence of the target nucleic acid.
- the present invention provides a use of the above-mentioned oligonucleotide pair, the above-mentioned double-stranded siRNA-like molecule, or the above-mentioned composition, for degrading or decreasing the level of a target nucleic acid, or for decreasing the production or the level of a polypeptide encoded by said target nucleic acid, in a cell, wherein the sense strand of the oligonucleotide pair comprises a nucleobase sequence substantially identical to a nucleobase sequence of the target nucleic acid.
- the present invention provides a use of the above-mentioned oligonucleotide pair, the above-mentioned double-stranded siRNA-like molecule, or the above-mentioned composition, for the preparation of a medicament, wherein the sense strand of the oligonucleotide pair comprises a nucleobase sequence substantially identical to a nucleobase sequence of the target nucleic acid.
- the present invention provides a use of the above-mentioned oligonucleotide pair, the above-mentioned double-stranded siRNA-like molecule, or the above-mentioned composition, for preventing or treating a disease or condition associated with the expression of a target nucleic acid, or of a polypeptide encoded by said target nucleic acid, in a subject, wherein the sense strand of the oligonucleotide pair comprises a nucleobase sequence substantially identical to a nucleobase sequence of the target nucleic acid.
- the present invention provides a use of the above-mentioned oligonucleotide pair, the above-mentioned double-stranded siRNA-like molecule, or the above-mentioned composition, for the preparation of a medicament for preventing or treating a disease or condition associated with the expression of a target nucleic acid, or of a polypeptide encoded by said target nucleic acid, in a subject, wherein the sense strand of the oligonucleotide pair comprises a nucleobase sequence substantially identical to a nucleobase sequence of the target nucleic acid.
- the present invention provides a use of the above-mentioned oligonucleotide pair, the above-mentioned double-stranded siRNA-like molecule, or the above-mentioned composition, as a medicament.
- the present invention provides a kit comprising the above-mentioned oligonucleotide pair, the above-mentioned double-stranded siRNA-like molecule, or the above-mentioned composition.
- the above-mentioned kit further comprises instructions for inhibiting the expression of a target nucleic acid in a cell, degrading or decreasing the level of the target nucleic acid, or for decreasing the production or the level of a polypeptide encoded by the target nucleic acid.
- FIG. 1 shows siRNA activity of 2′-fluorinated duplexes targeting nucleotides 1818-1836 of firefly luciferase.
- A Initial results (average of two transfections);
- B Confirmed activity of the most potent duplexes from (A), at lower concentrations (average of two transfections).
- FIG. 2 shows circular dichroism (CD) spectra of oligonucleotide duplexes jg1-jg15.
- A jg1-jg5, in which both strands have the same chemistry;
- B jg6-jg9, in which one of the two strands is a fully-modified chimeric strand;
- C jg10-jg13, in which one of the two strands is a fully-modified strand of a single chemistry; and
- D fully modified heteroduplexes jg14-jg15.
- the control duplex jg-1 double-strand RNA is included in all spectra for comparison.
- FIG. 4 shows the effect of small-interfering RNA (siRNA) transfections on eIF4E binding protein (4E-BP) 1 or 4E-BP2 expression.
- siRNA transfections were performed in HEK293T cells using Lipofectamine PlusTM reagent on cells plated at 70-80% confluence in a 24-well plate. For each well, either 2.5 ⁇ l (1) or 5 ⁇ l of siRNA duplex (20 ⁇ M annealed duplex) was mixed with 50 ⁇ l of OPTI-MEMTM and 1 ⁇ l of PlusTM reagent and incubated for 5 min. at room temperature (RT).
- RT room temperature
- FIG. 5 shows the effect of siRNA transfections on IFN production by HEK293T cells following stimulation with poly(I:C).
- siRNA transfections were performed in HEK293T cells using Lipofectamine PlusTM reagent on cells plated at 70-80% confluence in a 24-well plate.
- 5 ⁇ l of both 4E-BP1 and 4E-BP2 siRNA duplexes (modified H-611 or unmodified) (20 ⁇ M annealed duplex) (20 ⁇ M annealed duplex) were mixed with 75 ⁇ l of OPTI-MEMTM and 1 ⁇ l of PlusTM reagent and incubated for 5 min. at room temperature (RT).
- FIG. 6 shows luciferase knockdown experiments using oligonucleotide duplexes comprising a 2′F-ANA sense strands and antisense strands containing 2′F-ANA overhangs and LNA inserts;
- FIG. 7 shows luciferase knockdown experiments using oligonucleotide duplexes comprising a sense strand containing both 2′F-ANA and LNA;
- FIG. 8 shows luciferase knockdown experiments using oligonucleotide duplexes comprising a sense strand containing both 2′F-ANA and LNA annealed with a fully 2′F-RNA antisense strand;
- FIG. 9 shows c-myb knockdown experiments using 2′F-ANA/2′F-RNA/LNA siRNAs.
- A % gene expression relative to mock treatment following treatment with the indicated doses of various siRNA.
- B Survival rate of leukemia cells following siRNA treatment (y-axis represents number of leukemia cells still living after the indicated time periods after treatment with the indicated siRNA.
- the invention relates to oligonucleotides and their uses, for example in various types of gene silencing approaches.
- chemically-modified siRNA and more particularly oligonucleotide duplexes comprising one or more DNA-like and/or RNA-like nucleotides are able to mediate gene silencing.
- the present invention provides an oligonucleotide pair which can form a duplex, comprising:
- DNA-like residue refers to a conformation of for example a modified nucleoside or nucleotide which is similar to the conformation of a corresponding unmodified DNA unit.
- DNA-like conformation may be expressed for example as having a southern or eastern pseudorotation (P) value.
- DNA-like nucleotides include for example 2′-deoxyribonucleotides, 2′-deoxy-2′-substituted arabinonucleotides such as 2′-deoxy-2′-fluoroarabinonucleotides (2′F-ANA or FANA), and corresponding phosphorothioate analogs.
- RNA-like residue refers to a conformation of for example a modified nucleoside or nucleotide which is similar to the conformation of a corresponding unmodified RNA unit. RNA-like conformation may be expressed for example as having a northern P value. Further, RNA-like molecules tend to adopt an A-form helix while DNA-like molecules tend to adopt a B-form helix.
- RNA-like nucleotides include for example RNA nucleotides, 2′-substituted-RNA nucleotides such as 2′ Fluoro-RNA (2′F-RNA) nucleotides, locked nucleic acid (LNA) nucleotides (also defined as bridged nucleic acids or bicyclic nucleotides), 2′-fluoro-4′-thioarabinonucleotide (4′S-FANA nucleotides), 2′-O-alkyl-RNA and corresponding phosphorothioate analogs.
- 2′F-RNA 2′-substituted-RNA nucleotides
- LNA locked nucleic acid
- 4′S-FANA nucleotides 2′-fluoro-4′-thioarabinonucleotide
- RNA-like residues LNA and 2′F-RNA
- an oligonucleotide pair which can form a double-stranded duplex, for example:
- Sense DNA-like nucleotide(s), RNA-like nucleotide(s), or both
- Antisense DNA-like nucleotide(s), RNA-like nucleotide(s), or both
- Sense DNA-like nucleotide(s), RNA-like nucleotide(s), or both
- RNA-like nucleotide(s) RNA-like nucleotide(s)
- Antisense DNA-like nucleotide(s), RNA-like nucleotide(s), or both
- RNA-like nucleotide(s) RNA-like nucleotide(s)
- Antisense DNA-like nucleotide(s), RNA-like nucleotide(s), or both
- RNA-like nucleotide(s) RNA-like nucleotide(s)
- the present invention provides an oligonucleotide pair which can form a duplex comprising a sense (e.g., a first) strand and an antisense (e.g., a second) strand complementary to the sense (or first) strand, wherein the oligonucleotide duplex comprises:
- RNA 2′-substituted ribonucleotides
- LNA locked nucleic acid nucleotides
- the above-mentioned oligonucleotide duplex further comprises any combinations of DNA-like and/or RNA-like residues.
- Oligonucleotides of the invention may include those which contain intersugar backbone linkages such as phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages, phosphorothioates and those with formacetal (O—CH 2 —O), CH 2 —NH—O—CH 2 , CH 2 —N(CH 3 )—O—CH 2 (known as methylene(methylimino) or MMI backbone), CH 2 —O—N(CH 3 )—CH 2 , CH 2 —N(CH 3 )—N(CH 3 )—CH 2 and O—N(CH 3 )—CH 2 —CH 2 backbones (where phosphodiester is O—PO 2 —O—H 2 ).
- intersugar backbone linkages such as phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar link
- Oligonucleotides having morpholino backbone structures may also be used (U.S. Pat. No. 5,034,506).
- antisense oligonucleotides may have a peptide nucleic acid (PNA, sometimes referred to as “protein nucleic acid”) backbone, in which the phosphodiester backbone of the oligonucleotide may be replaced with a polyamide backbone wherein nucleosidic bases are bound directly or indirectly to aza nitrogen atoms or methylene groups in the polyamide backbone (Nielsen et al., Science 1991 254(5037): 1497-1500 and U.S. Pat. No. 5,539,082).
- the phosphodiester bonds may be substituted with structures which are chiral and enantiomerically specific. Persons of ordinary skill in the art will be able to select other linkages for use in practice of the invention.
- Nucleoside refers to a base-sugar combination, the base being attached to the sugar via an N-glycosidic linkage.
- Nucleotide refers to a nucleoside that additionally comprises a phosphate group attached to the sugar portion of the nucleoside.
- Base refers to a heterocyclic base moiety, which within a nucleoside or nucleotide is attached to the sugar portion thereof, generally at the 1′ position of the sugar moiety, also known as the anomeric position. This term includes both naturally-occurring and modified bases.
- the two most common classes of naturally-occurring bases are purines and pyrimidines, and comprise for example guanine, cytosine, thymine, adenine and uracil.
- a number of other naturally-occurring bases, as well as modified bases, are known in the art, for example, inosine, 5-methylcytosine, 2-thiothymine, 4-thiothymine, 7-deazaadenine, 9-deazaadenine, 3-deazaadenine, 7-deazaguanine, 9-deazaguanine, 6-thioguanine, isoguanine, 2,6-diaminopurine, hypoxanthine, and 6-thiohypoxanthine.
- Oligonucleotides of the invention may also include species which include at least one modified nucleotide base.
- purines and pyrimidines other than those normally found in nature may be used.
- modifications on the pentofuranosyl portion of the nucleotide subunits may also be effected. Examples of such modifications includes 2′-substitution/modification, such as 2′-O-alkyl- and 2′-halogen-substituted nucleotides.
- modifications at the 2′ position of sugar moieties which are useful in the present invention are OH, SH, SCH 3 , F, OCN, O(CH 2 ) n NH 2 or O(CH 2 ) n CH 3 where n is from 1 to about 10; C 1 to C 10 lower alkyl, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF 3 ; OCF 3 ; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH 3 ; SO 2 CH 3 ; ONO 2 ; NO 2 ; N 3 ; NH 2 ; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the pharmaco
- One or more pentofuranosyl groups may be replaced by another sugar, by an acyclic sugar, by a sugar mimic such as cyclobutyl or by another moiety which takes the place of the sugar such as the six carbon hexose, or the seven carbon oxapane.
- LNA generally refers to bicyclonucleotides and includes, for example, ⁇ -D, and ⁇ -L bicyclo nucleotides, bicyclo nucleotides such as xylo-locked nucleic acids (U.S. Pat. No. 7,084,125), L-ribo-locked nucleic acids (U.S. Pat. No. 7,053,207), 1′-2′ locked nucleic acids (U.S. Pat. Nos. 6,734,291 and 6,639,059), 3′-5′ locked nucleic acids (U.S. Pat. No. 6,083,482) as well as 2′-4′ locked nucleic acids.
- xylo-locked nucleic acids U.S. Pat. No. 7,084,125
- L-ribo-locked nucleic acids U.S. Pat. No. 7,053,207
- 1′-2′ locked nucleic acids U.S. Pat. Nos. 6,734,291 and 6,639,059
- the oligonucleotides in accordance with this invention may comprise from about 4 to about 100 nucleotide units, in further embodiments from about 10 to about 100, from about 4 to about 30, from about 10 to about 30, from about 18 to about 27, from about 19 to about 27, from about 18 to about 25, from about 19 to about 25, or from about 19 to about 23 nucleotide units, such as 19, 21 or 23 nucleotide units.
- a nucleotide unit is a base-sugar combination (or a combination of analogous structures) suitably bound to an adjacent nucleotide unit through phosphodiester or other bonds forming a backbone structure.
- heterocyclic base moiety of any nucleotides described herein may be one of the canonical bases of DNA or RNA, for example, adenine, cytosine, guanine, thymine or uracil.
- some of the heterocyclic base moieties may be made up of modified or non-canonical bases, for example, inosine, 5-methylcytosine, 2-thiothymine, 4-thiothymine, 7-deazaadenine, 9-deazaadenine, 3-deazaadenine, 7-deazaguanine, 9-deazaguanine, 6-thioguanine, isoguanine, 2,6-diaminopurine, hypoxanthine, and 6-thiohypoxanthine.
- the oligonucleotide comprises one or more of the following internucleotide linkages: a) phosphodiester linkages; b) phosphotriester linkages; c) phosphorothioate linkages; d) methylphosphonate linkages; e) boranophosphate linkages; or f) 2′,5′-phosphodiester linkages.
- the internucleotide linkages are phosphodiester linkages, phosphorothioate linkages or a combination thereof.
- the above-mentioned oligonucleotide pair or duplex comprises one or more 2′-substituted ANA and one or more 2′-substituted RNA (in one or both strands).
- the above-mentioned oligonucleotide pair or duplex comprises one or more 2′-substituted ANA and one or more LNA (in one or both strands).
- the above-mentioned oligonucleotide pair or duplex comprises one or more 2′-substituted ANA, one or more 2′-substituted RNA and one or more LNA (in one or both strands).
- each alternating segment comprises one residue (referred to as 1-1 altimer design or configuration).
- each alternating segment comprises two residues (referred to as 2-2 altimer design).
- each alternating segment comprises three residues (referred to as 3-3 altimer design).
- the above-mentioned alternating segments are in the sense strand.
- the above-mentioned oligonucleotide duplex comprises (in one or both strands) at least one 2′F-RNA residue.
- the above-mentioned 2′F-RNA residue is a 2′F-RNA pyrimidine.
- the above-mentioned at least one 2′F-RNA residue is in the antisense strand.
- the above-mentioned oligonucleotide pair or duplex is fully modified with one or more 2′F-RNA and 2′F-ANA residues.
- the above-mentioned oligonucleotide duplex comprises (on one or both strands) a combination of one or more 2′F-RNA pyrimidines and 2′F-ANA purines.
- the above-mentioned oligonucleotide duplex comprises (on one or both strands) one or more alternating segments of 2′F-RNA residues and 2′F-ANA residues (altimers), in a regular or irregular fashion.
- each segment comprises 1 to 5 residues.
- each segment comprises one residue (1-1 altimer design). In another embodiment, each segment comprises three residues (3-3 altimer design). In another embodiment, the above-mentioned oligonucleotide duplex comprises a mixture of 1-1 and 3-3 altimer designs. In another embodiment, the above-mentioned alternating segments of 2′F-RNA residues and 2′F-ANA residues (altimers) are on the sense strand.
- the above-mentioned oligonucleotide pair or duplex is fully modified with one or more 2′F-RNA residues, 2′F-ANA residues and LNA residues.
- the one or more LNA residues are on both the sense strand and the antisense strand.
- the one or more LNA residues are on the antisense strand.
- the one or more LNA residues are on the sense strand.
- the above-mentioned sense strand comprises (i) 2′F-ANA; (ii) 2′F-RNA; (iii) RNA; (iv) LNA; (v) DNA; or (vi) any combination of (i) to (v).
- the above-mentioned antisense strand comprises (i) 2′F-ANA; (ii) 2′F-RNA; (iii) RNA; (iv) LNA; (v) DNA; or (vi) any combination of (i) to (v).
- the above-mentioned sense strand comprises:
- the above-mentioned sense strand consists of:
- the above-mentioned antisense strand comprises:
- the above-mentioned antisense strand consists of:
- the above-mentioned oligonucleotide pair or duplex comprises:
- the sense strand in the case of a sense strand comprising a 19 residue core (with or without an additional overhang), the sense strand comprises LNA residues at positions 3, 11, 16 and/or 17. In a further embodiment, in the case of an antisense strand comprising a 19 residue core (with or without an additional overhang), the antisense strand comprises LNA residues at position 19 (as read from 5′ to 3′).
- the above-mentioned oligonucleotide pair or duplex comprises:
- Sense (2′F-RNA pyrimidines) x (2′F-ANA purines) y
- the above-mentioned oligonucleotide duplex comprises an overhang (e.g., a 5′ and/or 3′ overhang, on one strand or on both strands).
- the above-mentioned overhang is a 1 to 5 residues (e.g., nucleotides or modified nucleotides) overhang.
- the above-mentioned overhang is a 2 residues (e.g., nucleotides or modified nucleotides) overhang.
- a 19 residue sense and/or antisense strand may comprise an overhang of an additional 1 to 5 residues.
- a 2 residue overhang in both strands would result in sense and antisense strands of 21 residues each, 19 of which participate in base-pairing to form the duplex (the remaining 2 residues in each case representing the overhangs).
- the above-mentioned overhang comprises DNA, 2′F-ANA and/or 2′F-RNA residues. In a further embodiment, the above-mentioned overhang comprises two 2′F-ANA residues. In a further embodiment, the above-mentioned overhang comprising two 2′F-ANA residues is on the sense strand.
- the above-mentioned overhang comprises two 2′F-RNA residues. In a further embodiment, the above-mentioned overhang comprising two 2′F-RNA residues is on the antisense strand.
- the above-mentioned overhang is a 3′ overhang.
- the above-mentioned oligonucleotide pair or duplex is 5′ phosphorylated on one or both strands. In a further embodiment, the above-mentioned oligonucleotide pair or duplex is 5′ phosphorylated on the antisense strand.
- sequence e.g., nucleobase complementarity between the sense strand and the antisense strand, or the sequence (e.g., nucleobase) identity between the sense strand and a target nucleic acid (e.g., mRNA), or a portion thereof, may be “perfect” or “complete” (100% complementarity or identity).
- the complementarity between the sense strand and the antisense strand, or the identity between the sense strand and a target nucleic acid (e.g., mRNA), or a portion thereof), is substantial, for example greater than about 70%.
- a target nucleic acid e.g., mRNA
- one mismatch results in 94.7% complementarity
- two mismatches results in about 89.5% complementarily
- 3 mismatches results in about 84.2% complementarity
- 4 mismatches results in about 79% complementarity
- 5 mismatches results in about 74% complementarity.
- the sense strand has an identity of at least 12 nucleotides, in a further embodiment of at least 12 contiguous nucleotides, to at least a portion of a target nucleic acid (e.g., mRNA).
- the sense strand has an identity of at least 13 nucleotides, in further embodiments of at least 14, 15, 16, 17 or 18 nucleotides (contiguous or not), to at least a portion of a target nucleic acid.
- the sense strand has complete identity to a portion of a target mRNA, with the exception of overhanging nucleotides (3′ overhang).
- complementarity and identity refers to complementarity and identity of the nucleobase moieties (e.g., A, C, G, T or U), commonly referred to as “base pairing”, and is independent of for example modifications of the sugar moiety, such as those described herein.
- a guanine nucleoside residue having any sugar moiety i.e., modified or not
- Identity refers to sequence similarity between two peptides or two nucleic acid molecules. Identity can be determined by comparing each position in the aligned sequences. A degree of identity between nucleic acid or between amino acid sequences is a function of the number of identical or matching nucleotides or amino acids at positions shared by the sequences. As the term is used herein, a nucleic acid sequence is “substantially identical” to another sequence if the functional activity of the sequences is conserved. Two nucleic acid sequences are considered substantially identical if, when optimally aligned (with gaps permitted), they share at least about 70% sequence similarity or identity, or if the sequences share defined functional motifs.
- sequence similarity in optimally aligned substantially identical sequences may be at least 75%, 80%, 85%, 90% or 95%.
- An “unrelated” sequence shares less than 40% identity, though preferably less than about 25% identity, with a given reference sequence (e.g., a target nucleic acid).
- Substantially complementary nucleic acids are nucleic acids in which the complement of one molecule is substantially identical to the other molecule. Two nucleic acid or protein sequences are considered substantially identical if, when optimally aligned, they share at least about 70% sequence identity. In alternative embodiments, sequence identity may for example be at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. Optimal alignment of sequences for comparisons of identity may be conducted using a variety of algorithms, such as the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math 2: 482, the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443, the search for similarity method of Pearson and Lipman, 1988, Proc.
- the BLAST algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold.
- Initial neighbourhood word hits act as seeds for initiating searches to find longer HSPs.
- the word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction is halted when the following parameters are met: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
- the BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment.
- W word length
- B BLOSUM62 scoring matrix
- E expectation
- P(N) the smallest sum probability
- nucleotide or amino acid sequences are considered substantially identical if the smallest sum probability in a comparison of the test sequences is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
- hybridize to each other under moderately stringent, or preferably stringent, conditions Hybridization to filter-bound sequences under moderately stringent conditions may, for example, be performed in 0.5 M NaHPO 4 , 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.2 ⁇ SSC/0.1% SDS at 42° C. (see Ausubel, et al. (eds), 1989, Current Protocols in Molecular Biology, Vol. 1, Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York, at p. 2.10.3).
- hybridization to filter-bound sequences under stringent conditions may, for example, be performed in 0.5 M NaHPO 4 , 7% SDS, 1 mM EDTA at 65° C., and washing in 0.1 ⁇ SSC/0.1% SDS at 68° C. (see Ausubel, et al. (eds), 1989, supra).
- Hybridization conditions may be modified in accordance with known methods depending on the sequence of interest (see Tijssen, 1993, Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2 “Overview of principles of hybridization and the strategy of nucleic acid probe assays”, Elsevier, New York).
- stringent conditions are selected to be about 5° C. lower than the thermal melting point for the specific sequence at a defined ionic strength and pH.
- the sense strand and the antisense strand may be linked by a loop structure, which may be comprised of a non-nucleic acid polymer such as, inter alia, polyethylene glycol.
- the loop structure may be comprised of a nucleic acid, including modified and non-modified ribonucleotides and modified and non-modified deoxyribonucleotides.
- the 5′-terminus of the sense strand of the oligonucleotide duplex may be linked to the 3′-terminus of the antisense strand, or the 3 ‘-terminus of the sense strand may be linked to the 5 ’-terminus of the sense strand, said linkage being via a nucleic acid linker typically having a length between 2 to 100 nucleotides (or modified nucleotides), preferably about 2 to about 30 nucleobases.
- the above-mentioned oligonucleotide duplex is a hairpin duplex, that is a single strand comprising the sense and antisense strands which is self-complementary and folds back onto itself.
- the invention further provides a salt, such as a pharmaceutically acceptable salt, of any of the above-mentioned compounds (e.g., oligonucleotide, oligonucleotide duplex, siRNA or siRNA-like molecule) where applicable.
- a salt such as a pharmaceutically acceptable salt, of any of the above-mentioned compounds (e.g., oligonucleotide, oligonucleotide duplex, siRNA or siRNA-like molecule) where applicable.
- the present invention also relates to compounds which down-regulate expression of various genes, i.e., decrease production of an encoded polypeptide.
- the invention provides oligonucleotides/oligonucleotide duplexes of the invention and uses thereof in siRNA/RNAi applications, whereby expression of a nucleic acid encoding a polypeptide of interest, or a fragment thereof, may be inhibited or prevented using RNA interference (RNAi) technology, a type of post-transcriptional gene silencing.
- RNAi RNA interference
- RNAi may be used to create a pseudo “knockout”, i.e., a system in which the expression of the product encoded by a gene or coding region of interest is reduced, resulting in an overall reduction of the activity of the encoded product in a system.
- RNAi may be performed to target a nucleic acid of interest or fragment or variant thereof, to in turn reduce its expression and the level of activity of the product which it encodes.
- Such a system may be used for functional studies of the product, as well as to treat disorders related to the activity of such a product.
- RNAi is described in for example U.S. patent publications Nos. 2002/0173478 (Gewirtz; published Nov.
- RNAi Reagents and kits for performing RNAi are available commercially from for example Ambion Inc. (Austin, Tex., USA), New England Biolabs Inc. (Beverly, Mass., USA) and Invitrogen (Carlsbad, Calif., USA).
- RNAi The initial agent for RNAi in some systems is thought to be dsRNA or modified dsRNA molecules corresponding to a target nucleic acid.
- the dsRNA is then thought to be cleaved into short interfering RNAs (siRNAs) which are for example 21-23 nucleotides in length (19-21 by duplexes, each with 2 nucleotide 3′ overhangs).
- siRNAs short interfering RNAs
- the enzyme thought to effect this first cleavage step (the Drosophila version is referred to as “Dicer”) is categorized as a member of the RNase III family of dsRNA-specific ribonucleases.
- RNAi may be effected via directly introducing into the cell, or generating within the cell by introducing into the cell an siRNA or siRNA-like molecule or a suitable precursor (e.g., vector encoding precursor(s), etc.) thereof.
- An siRNA may then associate with other intracellular components to form an RNA-induced silencing complex (RISC).
- RISC RNA-induced silencing complex
- the RISC thus formed may subsequently target a transcript of interest via base-pairing interactions between its siRNA component and the target transcript by virtue of homology, resulting in the cleavage of the target transcript approximately 12 nucleotides from the 3′ end of the siRNA.
- RISC RNA-induced silencing complex
- RNAi may be effected by the introduction of suitable in vitro synthesized siRNA or siRNA-like molecules into cells. RNAi may for example be performed using chemically-synthesized RNA or modified RNA molecules. Alternatively, suitable expression vectors may be used to transcribe such RNA either in vitro or in vivo. In vitro transcription of sense and antisense strands (encoded by sequences present on the same vector or on separate vectors) may be effected using for example T7 RNA polymerase, in which case the vector may comprise a suitable coding sequence operably-linked to a T7 promoter. The in vitro-transcribed RNA may in embodiments be processed (e.g., using E.
- RNA duplex which is introduced into a target cell of interest.
- Other vectors may be used, which express small hairpin RNAs (shRNAs) which can be processed into siRNA-like molecules.
- shRNAs small hairpin RNAs
- Various vector-based methods have been described (see, e.g., Brummelkamp et al. [2002] Science 296: 550).
- Various methods for introducing such vectors into cells, either in vitro or in vivo are known in the art.
- a nucleic acid either a non-coding RNA (ncRNA) as well as an RNA encoding a polypeptide of interest (e.g. an mRNA), or a fragment thereof, may be inhibited by introducing into or generating within a cell an siRNA or siRNA-like molecule based on an oligonucleotide of the invention, corresponding to a nucleic acid of interest, or a fragment thereof, or to an nucleic acid homologous thereto (sometimes collectively referred to herein as a “target nucleic acid”).
- Target nucleic acid refers to a nucleic acid encoding a polypeptide (e.g., a coding RNA such as a mRNA), as well as to a non-coding nucleic acid, such as a non-coding RNA (ncRNA), i.e., an RNA that is not translated to a protein and which are involved in various cell functions including post-transcriptional modifications, gene regulation and propagation (virus).
- ncRNA include transfer RNA (tRNA), ribosomal RNA (rRNA) and small nuclear RNA (snRNA).
- siRNA-like molecule refers to a nucleic acid molecule similar to an siRNA (e.g., in size and structure) and capable of eliciting siRNA activity, i.e., to effect the RNAi-mediated inhibition of production of the polypeptide.
- a method may entail the direct administration of the siRNA or siRNA-like molecule into a cell.
- the siRNA or siRNA-like molecule is less than about 30 nucleotides in length. In a further embodiment, the siRNA or siRNA-like molecule is about 19-23 nucleotides in length.
- siRNA or siRNA-like molecule comprises a 19-21 by duplex portion, each strand having a 2 nucleotide 3′ overhang. In other embodiments, one or both strands may have blunt ends.
- the siRNA or siRNA-like molecule is substantially identical to a nucleic acid encoding a polypeptide of interest, or a fragment or variant (or a fragment of a variant) thereof. Such a variant is capable of encoding a protein having activity similar to the polypeptide of interest.
- the present invention further provides a double-stranded siRNA or siRNA-like molecule (or modified siRNA) comprising an oligonucleotide duplex of the invention.
- a long oligonucleotide (e.g., of about 80 to 500 nucleotides in length) comprising one or more stem and loop structures, where stem regions comprise the oligonucleotides of the invention, may be delivered in a carrier, preferably a pharmaceutically acceptable carrier, and may be processed intracellularly by endogenous cellular complexes to produce one or more smaller double stranded oligonucleotides (siRNA/siRNA-like molecules) of the present invention.
- This oligonucleotide is typically referred to as a tandem shRNA construct.
- the above-mentioned siRNA is a 25 to 30 nucleotides, which may be substrates for the Dicer endonuclease (Kim D.-M. et al. Nature Biotechnology, vol. 23, pp. 222-226 (2005)).
- the present invention also provides a composition (e.g., a pharmaceutical composition) comprising an oligonucleotide, oligonucleotide duplex or siRNA-like molecule of the invention, and an excipient or carrier, such as a biologically or pharmaceutically acceptable carrier or excipient.
- a composition e.g., a pharmaceutical composition
- such compositions include an oligonucleotide, oligonucleotide duplex or siRNA-like molecule of the invention in a therapeutically or prophylactically effective amount sufficient to treat a condition/disease associated with the expression (e.g., overexpression) of a target nucleic acid, and/or of a polypeptide encoded by a target nucleic acid.
- the therapeutic composition may be soluble in an aqueous solution at a physiologically acceptable pH.
- pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are physiologically compatible.
- the carrier is suitable for parenteral administration.
- the carrier can be suitable for intravenous, intraperitoneal, intramuscular, topical, sublingual or oral administration, or for administration by inhalation.
- Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.
- compositions typically must be sterile and stable under the conditions of manufacture and storage.
- the composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration.
- the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
- the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
- isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
- Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin.
- an oligonucleotide of the invention can be administered in a time release formulation, for example in a composition which includes a slow release polymer.
- the active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants and microencapsulated delivery systems.
- Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers (PLG). Many methods for the preparation of such formulations are patented or generally known to those skilled in the art.
- Sterile injectable solutions can be prepared by incorporating the active compound (e.g. an oligonucleotide, oligonucleotide duplex, siRNA, or siRNA-like molecule of the invention) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
- the active compound e.g. an oligonucleotide, oligonucleotide duplex, siRNA, or siRNA-like molecule of the invention
- dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
- the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
- an oligonucleotide of the invention may be formulated with one or more additional compounds that enhance its solubility.
- Suitable methods for siRNA delivery to effect RNAi include any method by which a siRNA can be introduced into an organelle, a cell, a tissue or an organism, as described herein or as would be known to one of ordinary skill in the art. Such methods include, but are not limited to, direct delivery of siRNA such as by injection including microinjection, electroporation, calcium phosphate precipitation, using DEAE-dextran followed by polyethylene glycol, direct sonic loading, liposome-mediated transfection, microprojectile bombardment, agitation with silicon carbide fibers, Agrobacterium -mediated transformation, PEG-mediated transformation, desiccation/inhibition-mediated uptake, and the like.
- direct delivery of siRNA such as by injection including microinjection, electroporation, calcium phosphate precipitation, using DEAE-dextran followed by polyethylene glycol, direct sonic loading, liposome-mediated transfection, microprojectile bombardment, agitation with silicon carbide fibers, Agrobacterium -mediated transformation, PEG-
- an organelle, cell, tissue or organism may be stably or transiently transformed.
- the oligonucleotide, double stranded molecule/duplex, siRNA molecule or composition of the invention may be delivered in liposome or lipofectin formulations and the like and are prepared by methods well known to those skilled in the art. Such methods are described, for example, in U.S. Pat. Nos. 5,593,972, 5,589,466, and 5,580,859.
- compositions of the present invention comprising an oligonucleotide duplex, siRNA, or siRNA-like molecule of the invention, may be provided in a kit or commercial package.
- the kit may further comprise instructions for the use of the oligonucleotide duplex, siRNA, or siRNA-like molecule for the inhibition of a target gene expression, and/or prevention and/or treatment of a disease/condition associated with expression (e.g., overexpression) of a target nucleic acid or gene.
- the kit may further comprise a validated positive control siRNA that targets a housekeeping gene and/or a validated negative control siRNA that is nontargeting.
- the kit may further comprise one or more reagents, such as reagents for introducing the oligonucleotide duplex, siRNA, or siRNA-like molecule of the invention into a cell (e.g., transfection/transformation reagents) and/or reagents for assessing knockdown of the intended target gene such as antibodies for monitoring knockdown at the protein level by immunofluorescence or Western analysis, reagents for assessing enzymatic activity or presence of a reporter protein, or reagents for assessing cell viability.
- RT-PCR primers and probes may be included for detection of target or reporter mRNA.
- the kit may further comprise a container (e.g., vial, test tube, flask, bottle, syringe or other packaging means) into which the oligonucleotide duplex, siRNA, or siRNA-like molecule may be placed/aliquoted, as well as devices for administering the oligonucleotide duplex, siRNA, or siRNA-like molecule to a subject (e.g., syringe).
- a container e.g., vial, test tube, flask, bottle, syringe or other packaging means
- a container e.g., vial, test tube, flask, bottle, syringe or other packaging means
- a subject e.g., syringe
- the invention further provides a method of inhibiting the expression of a target gene/nucleic acid, or of degrading or decreasing the level of a target gene/nucleic acid, in a biological system (e.g., a cell, a tissue, an organ, a subject), e.g., to inhibit production of a polypeptide encoded by the target gene/nucleic acid, comprising introducing into the system the above-mentioned oligonucleotide duplex, siRNA or siRNA-like molecule.
- a biological system e.g., a cell, a tissue, an organ, a subject
- a method of inhibiting production of the product of a gene (“gene silencing”; e.g., of a deleterious gene) in a patient in need thereof is provided.
- Gene silencing refers to an inhibition or reduction of the expression of the protein encoded by a particular nucleic acid sequence or gene (e.g., a deleterious gene).
- the method comprises administering to the patient a therapeutically effective amount of oligonucleotide, a double stranded molecule/duplex, an siRNA molecule or a composition of the invention.
- the target gene or nucleic acid is a viral, bacterial or mammalian (e.g., human) gene.
- the invention further provides a method of treating a condition associated with expression of a gene/nucleic acid in a subject, e.g., associated with the production of a polypeptide encoded by the target gene/nucleic acid, the method comprising administering the oligonucleotide duplex, siRNA or siRNA-like molecule to the subject (or to a cell, tissue, organ from the subject), wherein the siRNA or siRNA-like molecule is targeted to (or specific for) the gene/nucleic acid.
- the invention further provides a use of the siRNA or siRNA-like molecule for the preparation of a medicament.
- the invention further provides a use of the above-mentioned siRNA or siRNA-like molecule for a method selected from: (a) gene silencing; (b) inhibiting gene expression/polypeptide production in a biological system; (c) inhibiting gene expression/polypeptide production in a subject; (d) degrading or decreasing the level of a target gene/nucleic acid in a biological system or a subject; (d) treating a disease/condition associated with the production of a polypeptide encoded by a gene/nucleic acid in a subject; and (e) preparation of a medicament, for example a medicament for treating a disease or condition associated with expression (e.g., overexpression) of a nucleic acid/gene in a subject.
- a method selected from: (a) gene silencing; (b) inhibiting gene expression/polypeptide production in a biological system; (c) inhibiting gene expression/polypeptide production in a subject; (d) degrading or decreasing the
- an oligonucleotide pair, duplex, siRNA and/or siRNA-like molecule of the invention may be used prophylactically and/or therapeutically in formulations or medicaments to prevent or treat a disease/condition associated with the expression of a target nucleic acid or gene.
- the invention provides corresponding methods of medical treatment, in which a therapeutic dose of an oligonucleotide of the invention is administered in a pharmacologically acceptable formulation, e.g., to a patient or subject in need thereof.
- a “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as a reduction or reversal in progression of a disease associated with the production of a polypeptide encoded by a target nucleic acid or gene.
- a therapeutically effective amount of an oligonucleotide pair, duplex, siRNA and/or siRNA-like molecule of the invention may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response.
- a therapeutically effective amount is also one in which any toxic or detrimental effects of the compound are outweighed by the therapeutically beneficial effects.
- a “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as preventing or inhibiting the rate of onset or progression of a disease associated with the production of a polypeptide encoded by a target nucleic acid or gene.
- a prophylactically effective amount can be determined as described above for the therapeutically effective amount.
- specific dosage regimens may be adjusted over time according to the individual need and the professional judgement of the person administering or supervising the administration of the compositions.
- the invention further provides a use of an oligonucleotide, pair or duplex of the invention or the above-mentioned composition for degrading or decreasing the level of a target nucleic acid, or of decreasing the production or the level of a polypeptide encoded by a target nucleic acid or gene or for the prevention and/or treatment of a disease/condition associated with production of a polypeptide encoded by a target nucleic acid or gene.
- the invention further provides a use of an oligonucleotide of the invention for the preparation of a medicament.
- the medicament is for prevention and/or treatment of a disease or condition associated with expression (e.g., overexpression) of a target nucleic acid or gene.
- the target gene/nucleic acid can be a gene/nucleic acid derived from a cell, an endogenous gene, a transgene, or exogenous genes such as genes of a pathogen, for example, a virus, which is present in the cell after infection thereof.
- the cell having the target gene may be from the germ line or somatic, totipotent or pluripotent, dividing or non-dividing, parenchyma or epithelium, immortalized or transformed, or the like.
- the cell can be a gamete or an embryo; if an embryo, it can be a single cell embryo or a constituent cell or cells from a multicellular embryo.
- embryo thus encompasses fetal tissue.
- the cell having the target gene may be an undifferentiated cell, such as a stem cell, or a differentiated cell, such as from a cell of an organ or tissue, including fetal tissue, or any other cell present in an organism.
- Cell types that are differentiated include adipocytes, fibroblasts, myocytes, cardiomyocytes, endothelium, neurons, glia, blood cells, megakaryocytes, lymphocytes, macrophages, neutrophils, eosinophils, basophils, mast cells, leukocytes, granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts, hepatocytes, and cells, of the endocrine or exocrine glands.
- the oligonucleotide pair, duplex, siRNA or siRNA-like molecule of the invention may be associated with, for example, a cell-targeting ligand.
- a “cell targeting ligand” is a cell-directing molecule that has specificity for targeted sites such as cell surface receptors. This allows, for example, a more specific delivery of the oligonucleotide pair, duplex, siRNA or siRNA-like molecule to a particular cell/cell type, tissue or organ.
- the present invention provides a method for increasing/improving the efficacy, potency and/or stability (e.g., in vivo stability) of an oligonucleotide duplex, comprising incorporating into said duplex (a) one or more 2′-substituted arabinonucleotides (ANA); and (b) (i) one or more 2′-substituted ribonucleotides (RNA), (ii) one or more locked nucleic acid nucleotides (LNA), or (iii) a combination of (i) and (ii).
- ANA 2′-substituted arabinonucleotides
- RNA 2′-substituted ribonucleotides
- LNA locked nucleic acid nucleotides
- the present invention provides a method for reducing off-target effects of an oligonucleotide duplex, comprising incorporating into said duplex (a) one or more 2′-substituted arabinonucleotides (ANA); and (b) (i) one or more 2′-substituted ribonucleotides (RNA), (ii) one or more locked nucleic acid nucleotides (LNA), or (iii) a combination of (i) and (ii).
- ANA 2′-substituted arabinonucleotides
- RNA 2′-substituted ribonucleotides
- LNA locked nucleic acid nucleotides
- the invention further provides a method of synthesizing an oligonucleotide of the invention, the method comprising: (a) 5′-deblocking; (b) coupling; (c) capping; and (d) oxidation; wherein (a), (b), (c) and (d) are repeated under conditions suitable for the synthesis of the oligonucleotide, wherein the synthesis is carried out in the presence of a suitable nucleotide monomer described herein (e.g., RNA, DNA, 2′F-ANA, 2′F-RNA, LNA).
- a suitable nucleotide monomer described herein e.g., RNA, DNA, 2′F-ANA, 2′F-RNA, LNA.
- the invention further provides a method to prepare an oligonucleotide duplex of the invention comprising combining a first (e.g., sense) strand comprising an oligonucleotide of the invention and a second (e.g., antisense) strand substantially complementary to the first strand under conditions permitting formation of a duplex via base-pairing between the first and second strands.
- a first (e.g., sense) strand comprising an oligonucleotide of the invention and a second (e.g., antisense) strand substantially complementary to the first strand under conditions permitting formation of a duplex via base-pairing between the first and second strands.
- the synthesis is carried out on a solid phase, such as on a solid support selected from the group consisting of controlled pore glass, polystyrene, polyethylene glycol, polyvinyl, silica gel, silicon-based chips, cellulose paper, polyamide/kieselgur and polacryloylmorpholide.
- the monomers may be used for solution phase synthesis or ionic-liquid based synthesis of oligonucleotides.
- 5′-Deblocking refers to a step in oligonucleotide synthesis wherein a protecting group is removed from a previously added nucleoside (or a chemical group linked to a solid support), to produce a reactive hydroxyl which is capable of reacting with a nucleoside molecule, such as a nucleoside phosphoramidite or H-phosphonate.
- Protecting group refers to a moiety that is temporarily attached to a reactive chemical group to prevent the synthesis of undesired products during one or more stages of synthesis. Such a protecting group may then be removed to allow for step of the desired synthesis to proceed, or to generate the desired synthetic product.
- protecting groups are trityl (e.g., monomethoxytrityl, dimethoxytrityl), silyl, levulinyl and acetyl groups.
- “Coupling” as used herein refers to a step in oligonucleotide synthesis wherein a nucleoside is covalently attached to the terminal nucleoside residue of the oligonucleotide (or to the solid support via for example a suitable linker), for example via nucleophilic attack of an activated nucleoside phosphoramidite, H-phosphonate, phosphotriester, pyrophosphate, or phosphate in solution by a terminal 5′-hydroxyl group of a nucleotide or oligonucleotide bound to a support.
- Such activation may be effected by an activating reagent such as tetrazole, 5-ethylthio-tetrazole, 4,5-dicyanoimidazole (DCI), and/or pivaloyl chloride.
- Capping refers to a step in oligonucleotide synthesis wherein a chemical moiety is covalently attached to any free or unreacted hydroxyl groups on the support bound nucleic acid or oligonucleotide (or on a chemical linker attached to the support). Such capping is used to prevent the formation of for example sequences of shorter length than the desired sequence (e.g., containing deletions).
- An example of a reagent which may be used for such capping is acetic anhydride.
- the capping step may be performed either before or after the oxidation (see below) of the phosphite bond.
- Oxidation refers to a step in oligonucleotide synthesis wherein the newly synthesized phosphite triester or H-phosphonate diester bond is converted into pentavalent phosphate triester or diester bond.
- oxidation also refers to the addition of a sulfur atom to generate a phosphorothioate linkage.
- Oligonucleotide synthesis Standard conditions for solid-phase oligonucleotide synthesis were used for the synthesis of all oligonucleotides, at a 0.8 to 1.0 ⁇ mol scale. 4,5-Dicyanoimidazole (0.50 M in acetonitrile) or 5-ethylthiotetrazole (0.25 M in acetonitrile) were used as activators, and 0.10 M iodine in 1:2:10 pyridine:water:THF was used as oxidant (wait time during the oxidation step was 24 seconds). Phosphoramidites were prepared as 0.15 M solutions (RNA amidites) or 0.08-0.10 M solutions (DNA, 2′-fluoro amidites).
- 5′-phosphorylation of oligonucleotides was generally accomplished on the CPG solid support, by treating the newly-synthesized oligonucleotide with bis(2-cyanoethyl)-diisopropylaminophosphoramidite and ethylthiotetrazole, followed by normal deprotection conditions.
- ESI-MS was used to confirm the success of the phosphorylation reaction.
- CD spectra were obtained on a JascoTM J-720 spectropolarimeter at 20° C. using samples annealed in the same buffer and under the same conditions as for the thermal denaturation studies. Spectra were baseline-corrected with respect to a blank containing the buffer but no duplex. Smoothing and adjustment for duplex concentration were effected using the Spectra-Manager program (Jasco).
- siRNA assays luciferase inhibition. HeLa X1/5 cells that stably express firefly luciferase were grown as previously described (Wu, H. et al. J. Biol. Chem. 1999, 274: 28270-28278). The day prior to transfection, 0.5 ⁇ 10 5 cells were plated in each well of a 24-well plate. The next day, the cells were incubated with increasing amounts of siRNAs premixed with lipofectamine-plusTM reagent (Invitrogen) using 1 ⁇ L of lipofectamine and 4 ⁇ L of the plus reagent per 20 pmol of siRNA (for the highest concentration tested).
- lipofectamine-plusTM reagent Invitrogen
- each siRNA was diluted into dilution buffer (30 mM HEPES-KOH, pH 7.4, 100 mM KOAc, 2 mM MgOAc 2 ) and the amount of lipofectamine-plus reagent used relative to the siRNAs remained constant.
- the cells were lysed in hypotonic lysis buffer (15 mM K 3 PO 4 , 1 mM EDTA, 1% Triton, 2 mM NaF, 1 mg/ml BSA, 1 mM DTT, 100 mM NaCl, 4 ⁇ g/mL aprotinin, 2 ⁇ g/mL leupeptin and 2 ⁇ g/mL pepstatin) and the firefly light units were determined using a Fluostar Optima 96-well plate bioluminescence reader (BMG Labtech) using firefly substrate as described (Novac, O. et al. J. Nucl. Acids Res. 2004, 32: 902-915).
- hypotonic lysis buffer 15 mM K 3 PO 4 , 1 mM EDTA, 1% Triton, 2 mM NaF, 1 mg/ml BSA, 1 mM DTT, 100 mM NaCl, 4 ⁇ g/mL aprotinin, 2
- the luciferase counts were normalized to the protein concentration of the cell lysate as determined by the DC protein assay (BioRad). Error bars represent the standard deviation of at least four transfections. Cotransfecting the siRNAs and the plasmid pCl-hRL-con expressing the Renilla luciferase mRNA (Pillai, R. S. et al. Science 2005, 309: 1573-1576) in the same cell line showed no difference in expression of this reporter, demonstrating the specificity of the RNAi effects.
- IFN production using the HEK-BlueTM IFN detection assay 48 hours after siRNA transfection, cells were left untreated or treated with 1 ug/ml of poly(I:C) for 24 hours. The amount of IFN in the supernatant was measured according to the manufacturer's instructions (InvivoGen). Briefly, supernatants were mixed with HEK-BlueTM cells that carry a reporter gene expressing a secreted alkaline phosphatase under the control of the interferon stimulated response element 9 (ISRE9) promoter. In response to IFN exposure, the HEK-BlueTM cells release soluble alkaline phosphatase that is quantified by mixing the supernatant with Quanti BlueTM (InvivoGen) reagent and measuring the absorbance at 650 nm.
- ISRE9 interferon stimulated response element 9
- duplexes containing fully-modified 2′F-ANA and 2′F-RNA strands were made (Table I). These duplexes target positions 1818-1836 of the firefly luciferase gene (RefSeq accession number M15077).
- a series of chimeric strands containing both 2′-fluoro epimers was also designed.
- One chimera consisted of 2′F-RNA pyrimidines and 2′F-ANA purines.
- Another pair of strands was a “1-1 altimer” structure, with alternating 2′F-ANA and 2′F-RNA residues. For all of these 2′F-ANA/2′F-RNA chimeric strands, the 3′-overhang was always made of 2′F-ANA.
- RNAi activity of all duplexes was tested under the same conditions described above. Results are shown in FIG. 1 .
- duplexes jg-6, jg-8, jg-10 and jg-12 contained a modified sense strand paired with an RNA antisense strand.
- the best of these four duplexes is jg-6, containing a purine/pyrimidine chimeric sense strand.
- the second-best duplex is duplex jg-8, containing the 1-1 altimer configuration in the sense strand.
- RNAi activity of duplexes jg-6-jg-13 allows to evaluate the appropriateness of each type of modified strand architecture (2′F-ANA, 2′F-RNA, purine/pyrimidine and 1-1 altimer) in the sense or antisense strands.
- Sense/antisense preferences are observed for all four types of modified strands.
- Duplexes jg-6, jg-8 and jg-12 are more active than jg-7, jg-9 and jg-13, respectively, revealing that both chimeric constructs and the 2′F-ANA strand are better-tolerated in the sense strand than the antisense strand.
- duplexes jg-8 and jg-9 containing one 1-1 altimer strand jg-8 (1-1 altimer in the sense strand) was one of the most active duplexes tested, while jg-9 (1-1 altimer in the antisense strand) was inactive.
- FIG. 1 shows that jg-11 is more active than jg-10, thus suggesting that 2′F-RNA is better-tolerated in the antisense than the sense strand. It is believed that this is the first time a fully-modified or heavily-modified strand has been observed to be better tolerated in the antisense than the sense strand.
- a 2′F-ANA sense strand and a 2′F-RNA antisense strand formed a duplex that was found to be active as well. Indeed, synergy between these two modifications is observed in the case of duplex jg-14, which is more active than either of the duplexes jg-11 or jg-12 from which it is derived. On the other hand, reversing the sense/antisense combination gave jg-15, one of the least potent siRNAs tested in this study.
- the thermal stabilities of the duplexes were tested by heating the annealed duplexes, in physiological buffer, and measuring the change in the absorbance at 260 nm (A 260 ). Binding affinities of the modified duplexes vary widely. There was no correlation between RNAi activity and binding affinity. For example, two of the most active duplexes we tested were jg-4 and jg-8, with T m values of >90° C. and 48.2° C., respectively. The most potent duplex, the fully fluorinated heteroduplex jg-14, had a T m about 20° C. higher than that that of native RNA duplex (80.1° C. vs 61.8° C.).
- duplexes jg-1 and jg-4 feature a more strongly negative band at 210 nm. This is consistent with the degree of A-form helicity of the duplexes (Ratmeyer, L. et al. Biochemistry 1994, 33: 5298-5304).
- 2′F-RNA duplex jg-4 also has the highest intensity for its 270 nm band, followed by native RNA duplex jg-1, then the 2F-ANA-containing strands.
- Fully-2′F-ANA duplex jg-5 is quite B-form in character, as evidenced by the fact that its 270 nm band is of the lowest intensity and contains a shoulder above 280 nm, and its 245 nm negative band is significantly more negative than the other duplexes (Ratmeyer, L. et al. 1994, supra).
- duplexes jg-6-jg-13 For duplexes jg-6-jg-13, a modified sense strand corresponded to higher molar ellipticity at 220 nm than was observed for the native and antisense-modified duplexes. Thus, the intensity of the 220 nm band for the various sense antisense pairs jg-6/jg-7, jg-8/jg-9, jg-10/jg-11 and jg-12/jg-13 was always higher for the first member of each pair.
- duplex jg-14 and jg-15 in which both strands were modified, the more potent duplex jg-14 featured higher intensity for its 220 nm band, and indeed, in the whole range from 205-250 nm. It is not clear why such a large difference is observed between these two duplexes at lower wavelengths.
- Duplex jg-15 should have more A-form character since it has more strongly negative peaks at 210 nm, but the higher T m of jg-14 implies that it has more A-form character than jg-15 (Ratmeyer, L. et al. 1994, supra).
- RNA duplexes are presented in Table II.
- Each of two antisense strands (either RNA or 2′F-RNA) was paired with each of six modified sense strands (2′F-ANA or a 2′F-ANA-2′F-RNA chimera).
- the potency of these strands to induce RNAi was evaluated and the results are presented in FIG. 3 .
- duplexes can be thought of as belonging to two sub-series, the first with an RNA antisense strand (kl-1 to kl-6) and the second with a 2′F-RNA antisense strand (kl-7 to kl-12). Comparing the corresponding members of each series (kl-1 to kl-7, kl-2 to kl-8, etc), it is clear that all of the modified sense strands show better potency when paired to a 2′F-RNA antisense strand than an RNA antisense strand.
- the sense strands follow the same order, with either antisense strand.
- the “worst” sense strand is all 2′F-ANA (kl-2 and kl-8), followed by the “fr-type” sense strand containing five RNA inserts (kl-3 and kl-9). It should be noted, however, that both kl-8 and kl-9 are nonetheless more potent than the control.
- duplexes kl-7 and kl-11 seem to be silencing at their maximum efficacy, since the dose response is essentially flat.
- the chimeric sense strand of kl-11 thus allows higher efficacy silencing (relative luciferase level of 0.12-0.15 instead of 0.21-0.24).
- 2′F-ANA and 2′F-RNA can be combined in various ways in siRNA duplexes.
- two types of combinations of these two modifications lead to increased potency: combining both chemistries in the sense strand, and combining an 2′F-RNA antisense strand with a 2′F-ANA or chimeric sense strand. Examples of both of these types of synergistic combinations led to increased potency.
- siRNAs used in the 4E-BP inhibition studies described herein are provided in Table III.
- results presented at FIG. 4A indicate that unmodified siRNAs targeting human 4E-BP1 and 4E-BP2 are eliciting potent gene silencing (far right lanes in the two gels). As well, none of the scrambled (non-targeting) siRNAs affect expression levels of 4E-BP1 or 4E-BP2. Because Scrambled modified control 1 and 2 are chemically modified with 2′F-ANA and 2′F-RNA, these data indicate that the chemical modifications alone are not responsible for changes in expression of 4E-BP1 or 2.
- siRNAs comprising this modification are capable of silencing both 4E-BP1 and 2, although not as potently as the unmodified control after 24 hours, especially in the case of 4E-BP1.
- the — 611 modification architecture (alternating 2′F-ANA/2′F-RNA sense strand, fully 2′F-RNA antisense strand) appears to be more potent than — 14 in both cases, possibly even exceeding the potency of the unmodified control for 4E-BP2.
- LNA locked nucleic acid
- L-FL The first series, referred to as “L-FL”, were designed by combining 2′F-ANA sense strands with antisense strands containing 2′F-ANA overhangs and LNA inserts at positions previously observed to have RNAi activity.
- the sequences of the duplexes of the L-FL series are provided in Table IV.
- L-FL2 The second series, referred to as “L-FL2”, was designed based on 2′F-ANA/2′F-RNA architectures shown to have significant potency-improving synergy (see Examples 2 and 3 above).
- the sequences of the duplexes of the L-FL2 series are provided at Table V.
- siRNA labels SEQ ID NO: 5′- G C u U GAAG UC u UU AA u U AATT -3′ GD-21 L-FL2-1 43 5′- UUAAUUAAAGACUUCAAGCgg -3′ G1B 20 5′- G C u U GAAG UC u UU A A T u A A TT -3′ GD-22 L-FL2-2 44 5′- UUAAUUAAAGACUUCAAGCgg -3′ G1B 20 5′- G C u U GAAG UC u UU AA UU A A TT -3′ GD-23 L-FL2-3 45 5′- UUAAUUAAAGACUUCAAGCgg -3′ G1B 20 5′- G C u U GAAG UC u UU A A T U A A TT -3′ GD-24 L-FL2-4 46 5′- UUAAUUAAAGACUUCAA
- L-FL3 The third series, referred to as “L-FL3”, utilizes the same sense strands from L-FL2 annealed with all-2′F-RNA antisense strands.
- the sequences of the duplexes of the L-FL3 series are provided at Table VI.
- Each oligonucleotide was characterized by ESI-TOF mass spectroscopy (Table VII) and for some of the oligonucleotides by analytical denaturing PAGE followed by stains-all treatment.
- antisense strand GD2 containing two 3′ FANA (2′F-ANA) overhangs followed by a single LNA residue is compatible with the RNAi machinery, and in some cases can improve siRNA potency relative to a regular RNA antisense strand (compare L-FL1 with L-FL4).
- this antisense architecture was chosen to move forward with in further studies, now focused on probing for intrastrand 2′F-ANA/LNA synergy in the sense strand.
- potent gene silencing may be achieved using 2′F-ANA/2′F-RNA chimera siRNAs.
- Chimeric 2′F-ANA/LNA siRNA architectures comprising the L-FL2 series of siRNAs were then designed and studied.
- Sense strands were designed with alternating regions of 2′F-ANA and LNA moving from 5′ to 3′. LNA incorporation was kept to a minimum by surrounding strongly northern-puckered LNA inserts with RNA.
- Sense strands GD21-GD25 are identical until nucleotide 14, after which several patterns of chemical modification were employed.
- Strands GD21 and 22 feature alternating LNA-2′F-ANA regions designed to explore the effects of placing contrasting sugar puckers (northern vs. southeastern) side by side in a sense strand.
- GD23-25 feature various patterns of 2′F-ANA modification combined with unmodified RNA, including 1-1 altimer designs, 2-2 altimer designs, and fully RNA 3′ regions followed by 2′F-ANA overhangs.
- the T m of the oligonucleotide duplexes of the L-FL2 series is provided in Table VIII below.
- siRNAs were prepared by annealing GD21-GD25 with either a regular RNA antisense strand, or with GD2, the potent LNA/2′F-ANA antisense strand from the L-FL series. Indeed, despite the failure to introduce significant strand bias, several of these modified architectures were able to elicit potent gene silencing, comparable to or better than the native RISC substrate, dsRNA.
- the L-FL2 series demonstrates sense stand modification plans that are highly compatible with gene knockdown. However, in these cases the antisense strand remains unmodified, or only 3′-modified. It was next tested whether it was possible to combine these potent sense strand architectures with antisense strand modifications compatible with RISC, such as 2′F-RNA antisense strands.
- C-myb is a protooncogene implicated in leukemia. It encodes proteins essential for hematopoetic cell proliferation. 2′F-ANA/2′F-RNA and 2′F-ANA/2′F-RNA/LNA architectures shown to have luciferase and/or 4E-BP gene silencing activities were tested against another target, namely c-myb. The sequences of the duplexes of the C-myb series are provided in Table IX.
- siRNA labels SEQ ID NO: 5′- UGUUAUUGCCAAGCACUUAAA -3′ Cmyb-1 48 5′- UAAGUGCUUGGCAAUAACAGA -3′ 49 5′- TGT TGC G A T AAA -3′ Cmyb-2 50 5′- p -3′ 51 5′- T G u U ATTG CC a AG CA c U TAAA -3′ Cmyb-3 52 5′- p -3′ 51 5′- TGT TGC G A T AAA -3′ Cymb-4 50 5′- UAAGUGCUUGGCAAUAACAGA -3′ 49 5′- T G u U ATTG CC a AG CA c U TAAA -3′ Cmyb-5 52 5′- UAAGUGCUUGGCAAUAACAGA -3′ 49 5′- UGUUAUUGCCAAGCACUUAAA -3′ Cmyb-6 48 5′- UGUUAUUGCCAAGCACUUAAA -3′ Cmyb-6 48 5′- UGU
- 2′F-ANA/2′F-RNA and 2′F-ANA/2′F-RNA/LNA modified siRNA are capable of silencing gene expression in another target, and are better at silencing c-myb than unmodified siRNA at the lower dosages.
- the 2′F-ANA/2′F-RNA architecture appears to be more potent under the experimental conditions tested.
- FIG. 9B shows the survival rate (y-axis represents number of leukemia cells still living after the indicated time periods after treatment with siRNA designed to target c-myb and prevent leukemia cell proliferation) following siRNA treatment.
- unmodified siRNA-treated leukemia cells rebound 6 days after treatment and start proliferating again, whereas several of the modified siRNAs still prevent proliferation after 6 days. This suggests that modified siRNAs are not degraded as much as unmodified siRNAs after these time periods.
- novel chimeric siRNA architectures reported herein represent previously unexplored siRNA-mimics capable of equivalent or improved potencies compared to unmodified siRNA.
- siRNAs of Table II 5′ AACUCACCUGUGACCAAAAca Unmodified Control 1 5′ UUUUGGUCACAGGUGAGUUcc 4EBP-1 Human 2 5′ AAGACUCCAAAGUAGAAGUaa Unmodified Control 3 5′ ACUUCUACUUUGGAGUCUUca 4EBP-2 Human 4 and Murine 5′ AACUCACCUGUGGCCAAAAca Unmodified Control 5 5′ UUUUGGCCACAGGUGAGUUcc 4EBP-1 Murine 6 5′ AACTCACCTGTGGCCAAAACA 4EBP-1 Murine_14 7 5′ 8 5′ AACTCACCTGTGACCAAAACA 4EBP-1 Human_14 9 5′ 10 5′ AAGACTCCAAAGTAGAAGTAA 4EBP-2 Mouse_14 or 11 5′ 4EBP-2 Human_14 12 5′ AAC CCT G C A ACA 4EBP1 Mouse_611 13
Abstract
Description
- This application claims the benefit/priority of U.S. provisional application Ser. No. 61/059,186, filed on Jun. 5, 2008, of Canadian application serial No. 2,635,187, filed on Jun. 17, 2008 and of PCT application serial No. PCT/CA2008/002259, filed on Dec. 19, 2008. The contents of these applications are incorporated herein by reference in their entirety.
- This application contains a Sequence Listing in computer readable form entitled “11168—354—Seq listing”, created Jun. 5, 2009 having a size of 40.0 Ko, which is incorporated herein by reference.
- The invention relates to oligonucleotides, methods for their preparation and uses thereof, such as for decreasing the level of a target nucleic acid in a cell, and/or silencing the expression of a nucleic acid or gene of interest using small interfering RNA (siRNA) technologies.
- Gene silencing, i.e., selectively blocking the expression of a gene of interest, may be effected via the introduction of an antisense oligonucleotide (AON) or small interfering RNA (siRNA) into an organism (Uhlmann, E. and Peyman, A. Chem. Rev. 1990, 90: 543-84; Braasch, D. A. and Corey, D. R. Biochemistry 2002, 41: 4503-4510; Opalinska, J. B. and Gewirtz, A. M. Nat. Rev. Drug Discov. 2002, 1: 503-14; Dorsett, Y. and Tuschl, T. Nat. Rev. Drug Discov. 2004, 3: 318-329). Unfortunately, as with other nucleic acid-based drugs, siRNAs have poor serum stability, poor cellular uptake, and can elicit off-target and immunostimulatory side effects. Efforts to remedy these shortcomings have focused on the development of delivery vehicles for siRNAs, and on the development of chemically modified oligonucleotides with improved drug profiles.
- Much recent work has focussed on the chemical modification of siRNA. Dowler et al. (Dowler, T. et al. Nucl. Acids Res. 2006, 34: 1669-1675) were the first to show that 2′-deoxy-2′F -arabinonucleic acids (2′F-ANA) could be incorporated throughout the sense strand, including a fully-modified sense strand. Modification of the antisense-
strand 3′-overhang with 2′F-ANA brought a significant increase in potency, and several of the 2′F-ANA-modified duplexes have been able to surpass the native siRNA in potency. Furthermore, siRNA duplexes with extensive 2′F-ANA modification were found to have a significantly longer serum half-life than unmodified siRNAs. Modified siRNA duplexes containing 2′-fluoro-4′-thioarabinonucleotide (4′S-FANA) units were able to enter the RNAi pathway (Watts, J. K. et al. Nucl. Acids Res. 2007, 35: 1441-1451). One or two inserts internally in either strand gave duplexes of potency comparable to that of the control. The 4′S-FANA modification was also able to work with good efficiency in a duplex with a modified 2′F-ANA-RNA sense strand, demonstrating that 2′F-ANA (with its preference for southern and eastern conformations) can achieve synergy with 4′S-2′F-ANA (with its preference for northern conformations), in RNAi gene silencing. - 2′F-RNA is another siRNA modification, and partial 2′F-RNA modification is tolerated throughout both the sense and antisense strands, and some fully-modified 2′F-RNA siRNAs are also active. 2′F-RNA-modified siRNA duplexes have significantly increased serum stability (Layzer, J. M. et al. RNA, 2004, 10: 766-771). 2′F-RNA also increases the binding affinity of the duplex.
- An example of an increase in potency was observed for a fully modified siRNA made of a combination of 2′-O-Me and 2′F-RNA modified nucleotides, which was 500 times more potent than unmodified RNA (Allerson, C. R. et al. J. Med. Chem. 2005, 48: 901-904; Koller, E. et al. Nucl. Acids Res. 2006 34: 4467-4476). However, such a high degree of improvement was not observed for other sequences.
- These techniques present significant challenges, and there is a need for improvements in for example efficacy, in vivo stability and reduction of “off-target” effects (e.g., the silencing of a gene other than the intended target). There is therefore a continued need for improved oligonucleotide-based approaches.
- The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.
- The invention relates to oligonucleotides, methods for their preparation and uses thereof, such as for decreasing the level of a target nucleic acid in a cell, and/or silencing the expression of a nucleic acid or gene of interest using small interfering RNA (siRNA) technologies.
- In a first aspect, the present invention provides an oligonucleotide pair which can form a duplex, comprising:
-
- (a) a sense strand comprising (i) one or more DNA-like residues, (ii) one or more RNA-like residues, or (iii) both (i) and (ii); and
- (b) an antisense strand complementary to the sense strand, the antisense strand comprising (i) one or more DNA-like residues, (ii) one or more RNA-like residues, or (iii) both (i) and (ii).
- In a further aspect, the present invention provides an oligonucleotide pair which can form a duplex, comprising a sense strand and an antisense strand complementary to the sense strand, wherein the oligonucleotide pair comprises: (a) one or more 2′-substituted arabinonucleotides (ANA); and (b) (i) one or more 2′-substituted ribonucleotides (RNA), (ii) one or more locked nucleic acid nucleotides (LNA), or (iii) a combination of (i) and (ii).
- In an embodiment, the above-mentioned oligonucleotide pair comprises one or more 2′-substituted ANA and one or more 2′-substituted RNA. In another embodiment, the above-mentioned oligonucleotide pair comprises one or more 2′-substituted ANA and one or more LNA. In another embodiment, the above-mentioned oligonucleotide pair comprises one or more 2′-substituted ANA, one or more 2′-substituted RNA and one or more LNA.
- In an embodiment, the above-mentioned 2′-substitutent is an halogen. In a further embodiment, the above-mentioned halogen is fluorine (F).
- In an embodiment, the above-mentioned sense strand comprises: (i) 2′F-ANA only; (ii) 2′F-RNA only; (iii) a combination of 2′F-RNA and 2′F-ANA; (iv) RNA only; (v) a combination of 2′F-ANA and RNA; (vi) a combination of 2′F-ANA, RNA and LNA; or (vii) a combination of 2′F -ANA, 2′F-RNA and RNA.
- In an embodiment, the above-mentioned antisense strand comprises: (i) 2′F-RNA only; (ii) RNA only; (iii) 2′F-ANA only; (iv) a combination of 2′F-RNA and 2′F-ANA; (v) a combination of 2′F-ANA and RNA; (vi) a combination of 2′F-ANA, RNA and LNA; or (vii) a combination of 2′F-ANA, 2′F-RNA and RNA.
- In an embodiment, the above-mentioned sense strand and antisense strand have a length of 19 to 23 residues. In a further embodiment, the above-mentioned sense strand and antisense strand have a length of 21 residues.
- In another embodiment, the above-mentioned sense strand, antisense strand, or both, comprises an overhang at the 3′ end. In a further embodiment, the above-mentioned overhang is from 1 to 5 residues, in a
further embodiment 2 residues. - In an embodiment, the above-mentioned overhang comprises deoxyribonucleotides (DNA), 2′F-ANA, or a combination thereof.
- In an embodiment, the above-mentioned sense strand, antisense strand, or both, is/are phosphorylated at the 5′ end. In a further embodiment, the above-mentioned antisense strand is phosphorylated at the 5′ end.
- In another aspect, the present invention provides a double-stranded siRNA-like molecule comprising the above-mentioned oligonucleotide pair.
- In an embodiment, the above-mentioned sense and antisense strands are within an oligonucleotide of 15 to 80 nucleotides in length and such that the oligonucleotide or a portion thereof is capable of adopting an siRNA-like hairpin structure in which the sense and antisense strands form the stem of the hairpin structure.
- In another aspect, the present invention provides a composition comprising the above-mentioned oligonucleotide pair or the above-mentioned double-stranded siRNA-like molecule, and a pharmaceutically acceptable carrier.
- In another aspect, the present invention provides the above-mentioned oligonucleotide pair, the above-mentioned double-stranded siRNA-like molecule, or the above-mentioned composition, for decreasing the level of a target nucleic acid, or of a polypeptide encoded by said target nucleic acid, in a cell, wherein the sense strand of the oligonucleotide pair comprises a nucleobase sequence substantially identical to a nucleobase sequence of the target nucleic acid.
- In another aspect, the present invention provides the above-mentioned oligonucleotide pair, the above-mentioned double-stranded siRNA-like molecule, or the above-mentioned composition, for preventing or treating a disease or condition associated with the expression of a target nucleic acid, or of a polypeptide encoded by said target nucleic acid, in a subject, wherein the sense strand of the oligonucleotide pair comprises a nucleobase sequence substantially identical to a nucleobase sequence of the target nucleic acid.
- In another aspect, the present invention provides a method of degrading or decreasing the level of a target nucleic acid, or of decreasing the production or the level of a polypeptide encoded by said target nucleic acid, in a cell, the method comprising contacting the cell with the above-mentioned oligonucleotide pair, the above-mentioned double-stranded siRNA-like molecule, or the above-mentioned composition, wherein the sense strand of the oligonucleotide pair comprises a nucleobase sequence substantially identical to a nucleobase sequence of the target nucleic acid.
- In another aspect, the present invention provides a method of preventing or treating a disease or condition associated with the expression of a target nucleic acid, or of a polypeptide encoded by said target nucleic acid, in a subject, the method comprising administering to the subject an effective amount of the above-mentioned oligonucleotide pair, the above-mentioned double-stranded siRNA-like molecule, or the above-mentioned composition, wherein the sense strand of the oligonucleotide pair comprises a nucleobase sequence substantially identical to a nucleobase sequence of the target nucleic acid.
- In another aspect, the present invention provides a use of the above-mentioned oligonucleotide pair, the above-mentioned double-stranded siRNA-like molecule, or the above-mentioned composition, for degrading or decreasing the level of a target nucleic acid, or for decreasing the production or the level of a polypeptide encoded by said target nucleic acid, in a cell, wherein the sense strand of the oligonucleotide pair comprises a nucleobase sequence substantially identical to a nucleobase sequence of the target nucleic acid.
- In another aspect, the present invention provides a use of the above-mentioned oligonucleotide pair, the above-mentioned double-stranded siRNA-like molecule, or the above-mentioned composition, for the preparation of a medicament, wherein the sense strand of the oligonucleotide pair comprises a nucleobase sequence substantially identical to a nucleobase sequence of the target nucleic acid.
- In another aspect, the present invention provides a use of the above-mentioned oligonucleotide pair, the above-mentioned double-stranded siRNA-like molecule, or the above-mentioned composition, for preventing or treating a disease or condition associated with the expression of a target nucleic acid, or of a polypeptide encoded by said target nucleic acid, in a subject, wherein the sense strand of the oligonucleotide pair comprises a nucleobase sequence substantially identical to a nucleobase sequence of the target nucleic acid.
- In another aspect, the present invention provides a use of the above-mentioned oligonucleotide pair, the above-mentioned double-stranded siRNA-like molecule, or the above-mentioned composition, for the preparation of a medicament for preventing or treating a disease or condition associated with the expression of a target nucleic acid, or of a polypeptide encoded by said target nucleic acid, in a subject, wherein the sense strand of the oligonucleotide pair comprises a nucleobase sequence substantially identical to a nucleobase sequence of the target nucleic acid.
- In another aspect, the present invention provides a use of the above-mentioned oligonucleotide pair, the above-mentioned double-stranded siRNA-like molecule, or the above-mentioned composition, as a medicament.
- In another aspect, the present invention provides a kit comprising the above-mentioned oligonucleotide pair, the above-mentioned double-stranded siRNA-like molecule, or the above-mentioned composition. In an embodiment, the above-mentioned kit further comprises instructions for inhibiting the expression of a target nucleic acid in a cell, degrading or decreasing the level of the target nucleic acid, or for decreasing the production or the level of a polypeptide encoded by the target nucleic acid.
- Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.
- In the appended drawings:
-
FIG. 1 shows siRNA activity of 2′-fluorinated duplexes targeting nucleotides 1818-1836 of firefly luciferase. (A) Initial results (average of two transfections); (B) Confirmed activity of the most potent duplexes from (A), at lower concentrations (average of two transfections). In (A): black bars=2 nM, grey bars=10 nM and white bars=40 nM. In (B) black bars=0.4 nM, grey bars=2 nM and white bars=10 nM; -
FIG. 2 shows circular dichroism (CD) spectra of oligonucleotide duplexes jg1-jg15. (A) jg1-jg5, in which both strands have the same chemistry; (B) jg6-jg9, in which one of the two strands is a fully-modified chimeric strand; (C) jg10-jg13, in which one of the two strands is a fully-modified strand of a single chemistry; and (D) fully modified heteroduplexes jg14-jg15. The control duplex jg-1 (double-strand RNA) is included in all spectra for comparison. In (A) black line=jg-1, dashed line=jg-2, thin grey line=jg-3, thick grey line=jg-4, dotted line=jg-5. In (B) black line=jg-1, thick grey line=jg-6, thin grey line=jg-7, dotted line=jg-8, dashed line=jg-9. In (C) thick black line=jg-1, dashed line=jg-10, thin black line=jg-11, thick grey line=jg-12, dotted line=jg-13. In (D) black line=jg-1, dotted line=jg-14, dashed line=jg-15; -
FIG. 3 shows siRNA activity of 2′-fluorinated duplexes targeting nucleotides 515 to 533 of firefly luciferase. Black bars=40 nM, grey bars=10 nM and white bars=2 nM; -
FIG. 4 shows the effect of small-interfering RNA (siRNA) transfections on eIF4E binding protein (4E-BP) 1 or 4E-BP2 expression. (A) siRNA transfections were performed in HEK293T cells using Lipofectamine Plus™ reagent on cells plated at 70-80% confluence in a 24-well plate. For each well, either 2.5 μl (1) or 5 μl of siRNA duplex (20 μM annealed duplex) was mixed with 50 μl of OPTI-MEM™ and 1 μl of Plus™ reagent and incubated for 5 min. at room temperature (RT). A mixture of 4 μl of Lipofectamine™ reagent and 50 μl of OPTI-MEM™ was then added to the precomplexed RNA mix and incubated for 20 min. at RT before adding to cells. Five hours later, the transfection medium was replaced by complete medium. Cells were harvested 48 hours after transfection and proteins were extracted for analysis by Western blotting using antibodies against 4E-BP1, 4E-BP2 or β-actin. (B) Sequences of the siRNAs used in (A) (also shown in Table X below); -
FIG. 5 shows the effect of siRNA transfections on IFN production by HEK293T cells following stimulation with poly(I:C). siRNA transfections were performed in HEK293T cells using Lipofectamine Plus™ reagent on cells plated at 70-80% confluence in a 24-well plate. For each well, 5 μl of both 4E-BP1 and 4E-BP2 siRNA duplexes (modified H-611 or unmodified) (20 μM annealed duplex) were mixed with 75 μl of OPTI-MEM™ and 1 μl of Plus™ reagent and incubated for 5 min. at room temperature (RT). A mixture of 5 μl of Lipofectamine™ reagent and 75 μl of OPTI-MEM™ was then added to the precomplexed RNA mix and incubated for 20 min. at RT before adding to cells. Five hours later, the transfection medium was replaced by complete medium. 48 hours after transfection, cells were either left untreated or treated with 1 μg/ml of poly(I:C) for 24 hours. Supernatants from untreated and treated cells were collected and the amount of IFN was quantified using the HEK-Blue™ IFN-α/β Cells (InvivoGen, San Diego, USA) according to the manufacturer's protocol; -
FIG. 6 shows luciferase knockdown experiments using oligonucleotide duplexes comprising a 2′F-ANA sense strands and antisense strands containing 2′F-ANA overhangs and LNA inserts; -
FIG. 7 shows luciferase knockdown experiments using oligonucleotide duplexes comprising a sense strand containing both 2′F-ANA and LNA; -
FIG. 8 shows luciferase knockdown experiments using oligonucleotide duplexes comprising a sense strand containing both 2′F-ANA and LNA annealed with a fully 2′F-RNA antisense strand; -
FIG. 9 shows c-myb knockdown experiments using 2′F-ANA/2′F-RNA/LNA siRNAs. (A) % gene expression relative to mock treatment following treatment with the indicated doses of various siRNA. (B) Survival rate of leukemia cells following siRNA treatment (y-axis represents number of leukemia cells still living after the indicated time periods after treatment with the indicated siRNA. - The invention relates to oligonucleotides and their uses, for example in various types of gene silencing approaches. In the studies described herein, the inventors have shown that chemically-modified siRNA, and more particularly oligonucleotide duplexes comprising one or more DNA-like and/or RNA-like nucleotides are able to mediate gene silencing.
- Accordingly, in a first aspect, the present invention provides an oligonucleotide pair which can form a duplex, comprising:
-
- (a) a sense strand comprising (i) one or more DNA-like residues, (ii) one or more RNA-like residues, or (iii) both (i) and (ii); and
- (b) an antisense strand complementary to the sense strand, the antisense strand comprising (i) one or more DNA-like residues, (ii) one or more RNA-like residues, or (iii) both (i) and (ii).
- “DNA-like residue” as used herein in reference to conformation refers to a conformation of for example a modified nucleoside or nucleotide which is similar to the conformation of a corresponding unmodified DNA unit. DNA-like conformation may be expressed for example as having a southern or eastern pseudorotation (P) value. DNA-like nucleotides include for example 2′-deoxyribonucleotides, 2′-deoxy-2′-substituted arabinonucleotides such as 2′-deoxy-2′-fluoroarabinonucleotides (2′F-ANA or FANA), and corresponding phosphorothioate analogs. “RNA-like residue” as used herein in reference to conformation refers to a conformation of for example a modified nucleoside or nucleotide which is similar to the conformation of a corresponding unmodified RNA unit. RNA-like conformation may be expressed for example as having a northern P value. Further, RNA-like molecules tend to adopt an A-form helix while DNA-like molecules tend to adopt a B-form helix. RNA-like nucleotides include for example RNA nucleotides, 2′-substituted-RNA nucleotides such as 2′ Fluoro-RNA (2′F-RNA) nucleotides, locked nucleic acid (LNA) nucleotides (also defined as bridged nucleic acids or bicyclic nucleotides), 2′-fluoro-4′-thioarabinonucleotide (4′S-FANA nucleotides), 2′-O-alkyl-RNA and corresponding phosphorothioate analogs.
- The structure of a representative DNA-like residue (2′F-ANA) is illustrated below:
- The structures of examples of RNA-like residues (RNA, LNA and 2′F-RNA) are illustrated below:
- In a further aspect of the invention, an oligonucleotide pair is provided which can form a double-stranded duplex, for example:
- Sense: DNA-like nucleotide(s), RNA-like nucleotide(s), or both
- Antisense: DNA-like nucleotide(s), RNA-like nucleotide(s), or both
- Sense: DNA-like nucleotide(s), RNA-like nucleotide(s), or both
- Antisense: RNA-like nucleotide(s)
- Sense: DNA-like nucleotide(s)
- Antisense: DNA-like nucleotide(s), RNA-like nucleotide(s), or both
- Sense: RNA-like nucleotide(s)
- Antisense: DNA-like nucleotide(s), RNA-like nucleotide(s), or both
- Sense: DNA-like nucleotide(s)
- Antisense: RNA-like nucleotide(s)
- In another aspect, the present invention provides an oligonucleotide pair which can form a duplex comprising a sense (e.g., a first) strand and an antisense (e.g., a second) strand complementary to the sense (or first) strand, wherein the oligonucleotide duplex comprises:
- (a) one or more 2′-substituted arabinonucleotides (ANA); and
- (b) (i) one or more 2′-substituted ribonucleotides (RNA), (ii) one or more locked nucleic acid nucleotides (LNA), or (iii) a combination of (i) and (ii).
- In an embodiment, the above-mentioned oligonucleotide duplex further comprises any combinations of DNA-like and/or RNA-like residues.
- Oligonucleotides of the invention may include those which contain intersugar backbone linkages such as phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages, phosphorothioates and those with formacetal (O—CH2—O), CH2—NH—O—CH2, CH2—N(CH3)—O—CH2 (known as methylene(methylimino) or MMI backbone), CH2—O—N(CH3)—CH2, CH2—N(CH3)—N(CH3)—CH2 and O—N(CH3)—CH2—CH2 backbones (where phosphodiester is O—PO2—O—H2). Oligonucleotides having morpholino backbone structures may also be used (U.S. Pat. No. 5,034,506). In alternative embodiments, antisense oligonucleotides may have a peptide nucleic acid (PNA, sometimes referred to as “protein nucleic acid”) backbone, in which the phosphodiester backbone of the oligonucleotide may be replaced with a polyamide backbone wherein nucleosidic bases are bound directly or indirectly to aza nitrogen atoms or methylene groups in the polyamide backbone (Nielsen et al., Science 1991 254(5037): 1497-1500 and U.S. Pat. No. 5,539,082). The phosphodiester bonds may be substituted with structures which are chiral and enantiomerically specific. Persons of ordinary skill in the art will be able to select other linkages for use in practice of the invention.
- “Nucleoside” refers to a base-sugar combination, the base being attached to the sugar via an N-glycosidic linkage. “Nucleotide” refers to a nucleoside that additionally comprises a phosphate group attached to the sugar portion of the nucleoside. “Base”, “nucleic acid base” or “nucleobase” refer to a heterocyclic base moiety, which within a nucleoside or nucleotide is attached to the sugar portion thereof, generally at the 1′ position of the sugar moiety, also known as the anomeric position. This term includes both naturally-occurring and modified bases. The two most common classes of naturally-occurring bases are purines and pyrimidines, and comprise for example guanine, cytosine, thymine, adenine and uracil. A number of other naturally-occurring bases, as well as modified bases, are known in the art, for example, inosine, 5-methylcytosine, 2-thiothymine, 4-thiothymine, 7-deazaadenine, 9-deazaadenine, 3-deazaadenine, 7-deazaguanine, 9-deazaguanine, 6-thioguanine, isoguanine, 2,6-diaminopurine, hypoxanthine, and 6-thiohypoxanthine.
- Oligonucleotides of the invention may also include species which include at least one modified nucleotide base. Thus, purines and pyrimidines other than those normally found in nature may be used. Similarly, modifications on the pentofuranosyl portion of the nucleotide subunits may also be effected. Examples of such modifications includes 2′-substitution/modification, such as 2′-O-alkyl- and 2′-halogen-substituted nucleotides. Some specific examples of modifications at the 2′ position of sugar moieties which are useful in the present invention are OH, SH, SCH3, F, OCN, O(CH2)n NH2 or O(CH2)n CH3 where n is from 1 to about 10; C1 to C10 lower alkyl, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF3 ; OCF3; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH3; SO2 CH3; ONO2 ; NO2 ; N3; NH2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the pharmacodynamic properties of an oligonucleotide and other substituents having similar properties. One or more pentofuranosyl groups may be replaced by another sugar, by an acyclic sugar, by a sugar mimic such as cyclobutyl or by another moiety which takes the place of the sugar such as the six carbon hexose, or the seven carbon oxapane.
- LNA generally refers to bicyclonucleotides and includes, for example, β-D, and α-L bicyclo nucleotides, bicyclo nucleotides such as xylo-locked nucleic acids (U.S. Pat. No. 7,084,125), L-ribo-locked nucleic acids (U.S. Pat. No. 7,053,207), 1′-2′ locked nucleic acids (U.S. Pat. Nos. 6,734,291 and 6,639,059), 3′-5′ locked nucleic acids (U.S. Pat. No. 6,083,482) as well as 2′-4′ locked nucleic acids.
- In some embodiments, the oligonucleotides in accordance with this invention may comprise from about 4 to about 100 nucleotide units, in further embodiments from about 10 to about 100, from about 4 to about 30, from about 10 to about 30, from about 18 to about 27, from about 19 to about 27, from about 18 to about 25, from about 19 to about 25, or from about 19 to about 23 nucleotide units, such as 19, 21 or 23 nucleotide units. As will be appreciated, a nucleotide unit is a base-sugar combination (or a combination of analogous structures) suitably bound to an adjacent nucleotide unit through phosphodiester or other bonds forming a backbone structure.
- The heterocyclic base moiety of any nucleotides described herein may be one of the canonical bases of DNA or RNA, for example, adenine, cytosine, guanine, thymine or uracil. In other embodiments of the invention, some of the heterocyclic base moieties may be made up of modified or non-canonical bases, for example, inosine, 5-methylcytosine, 2-thiothymine, 4-thiothymine, 7-deazaadenine, 9-deazaadenine, 3-deazaadenine, 7-deazaguanine, 9-deazaguanine, 6-thioguanine, isoguanine, 2,6-diaminopurine, hypoxanthine, and 6-thiohypoxanthine.
- In other embodiments of the invention, the oligonucleotide comprises one or more of the following internucleotide linkages: a) phosphodiester linkages; b) phosphotriester linkages; c) phosphorothioate linkages; d) methylphosphonate linkages; e) boranophosphate linkages; or f) 2′,5′-phosphodiester linkages. In embodiments, the internucleotide linkages are phosphodiester linkages, phosphorothioate linkages or a combination thereof.
- In an embodiment, the above-mentioned oligonucleotide pair or duplex comprises one or more 2′-substituted ANA and one or more 2′-substituted RNA (in one or both strands).
- In another embodiment, the above-mentioned oligonucleotide pair or duplex comprises one or more 2′-substituted ANA and one or more LNA (in one or both strands).
- In another embodiment, the above-mentioned oligonucleotide pair or duplex comprises one or more 2′-substituted ANA, one or more 2′-substituted RNA and one or more LNA (in one or both strands).
- In embodiments, the DNA-like and RNA-like residues are in alternating segments within a strand, such as in an irregular fashion (whereby there may be differences in the number of residues per segment) or a regular fashion (whereby each segment has the same number of residues), or combinations thereof. In an embodiment, each alternating segment comprises one residue (referred to as 1-1 altimer design or configuration). In another embodiment, each alternating segment comprises two residues (referred to as 2-2 altimer design). In another embodiment, each alternating segment comprises three residues (referred to as 3-3 altimer design). In yet another embodiment, the above-mentioned alternating segments are in the sense strand.
- In an embodiment, the above-mentioned oligonucleotide duplex comprises (in one or both strands) at least one 2′F-RNA residue. In a further embodiment, the above-mentioned 2′F-RNA residue is a 2′F-RNA pyrimidine. In another embodiment, the above-mentioned at least one 2′F-RNA residue is in the antisense strand.
- In a further embodiment, the above-mentioned oligonucleotide pair or duplex is fully modified with one or more 2′F-RNA and 2′F-ANA residues. In another embodiment, the above-mentioned oligonucleotide duplex comprises (on one or both strands) a combination of one or more 2′F-RNA pyrimidines and 2′F-ANA purines. In another embodiment, the above-mentioned oligonucleotide duplex comprises (on one or both strands) one or more alternating segments of 2′F-RNA residues and 2′F-ANA residues (altimers), in a regular or irregular fashion. In a further embodiment, each segment comprises 1 to 5 residues. In a further embodiment, each segment comprises one residue (1-1 altimer design). In another embodiment, each segment comprises three residues (3-3 altimer design). In another embodiment, the above-mentioned oligonucleotide duplex comprises a mixture of 1-1 and 3-3 altimer designs. In another embodiment, the above-mentioned alternating segments of 2′F-RNA residues and 2′F-ANA residues (altimers) are on the sense strand.
- In another embodiment, the above-mentioned oligonucleotide pair or duplex is fully modified with one or more 2′F-RNA residues, 2′F-ANA residues and LNA residues. In an embodiment, the one or more LNA residues are on both the sense strand and the antisense strand. In another embodiment, the one or more LNA residues are on the antisense strand. In another embodiment, the one or more LNA residues are on the sense strand.
- In an embodiment, the above-mentioned sense strand comprises (i) 2′F-ANA; (ii) 2′F-RNA; (iii) RNA; (iv) LNA; (v) DNA; or (vi) any combination of (i) to (v).
- In an embodiment, the above-mentioned antisense strand comprises (i) 2′F-ANA; (ii) 2′F-RNA; (iii) RNA; (iv) LNA; (v) DNA; or (vi) any combination of (i) to (v).
- In an embodiment, the above-mentioned sense strand comprises:
-
- (i) 2′F-ANA only;
- (ii) 2′F-RNA only;
- (iii) a combination of 2′F-RNA and 2′F-ANA;
- (iv) RNA only;
- (v) a combination of 2′F-ANA and RNA;
- (vi) a combination of 2′F-ANA, 2′F-RNA, and RNA;
- (vii) a combination of 2′F-ANA, RNA and LNA.
- In a further embodiment, the above-mentioned sense strand consists of:
-
- (i) 2′F-ANA only;
- (ii) 2′F-RNA only;
- (iii) a combination of 2′F-RNA and 2′F-ANA;
- (iv) RNA only;
- (v) a combination of 2′F-ANA and RNA;
- (vi) a combination of 2′F-ANA, 2′F-RNA, and RNA: or
- (vii) a combination of 2′F-ANA, RNA and LNA.
- In another embodiment, the above-mentioned antisense strand comprises:
-
- (i) 2′F-RNA only;
- (ii) RNA only;
- (iii) 2′F-ANA only;
- (iv) a combination of 2′F-RNA and 2′F-ANA;
- (v) a combination of RNA and LNA; or
- (vi) a combination of 2′F-ANA, RNA and LNA.
- In a further embodiment, the above-mentioned antisense strand consists of:
-
- (i) 2′F-RNA only;
- (ii) RNA only;
- (iii) 2′F-ANA only;
- (iv) a combination of 2′F-RNA and 2′F-ANA;
- (v) a combination of RNA and LNA; or
- (vi) a combination of 2′F-ANA, RNA and LNA.
- In another embodiment, the above-mentioned oligonucleotide pair or duplex comprises:
-
- (a) Sense: a combination of 2′F-RNA and 2′F-ANA
- Antisense: RNA only;
- (b) Sense: a combination of 2′F-RNA pyrimidines and 2′F-ANA purines
- Antisense: RNA only;
- (c) Sense: a combination of 2′F-RNA and 2′F-ANA in a 1-1 altimer design
- Antisense: RNA only;
- (d) Sense: 2′F-ANA only
- Antisense: 2′F-RNA only;
- (e) Sense: a combination of 2′F-RNA and 2′F-ANA in a 3-3 altimer design
- Antisense: RNA only;
- (f) Sense: a combination of 2′F-RNA+2′F-ANA in 3-3 and 1-1 altimer designs
- Antisense: RNA only;
- (g) Sense: a combination of 2′F-ANA and RNA
- Antisense: 2′F-RNA only;
- (h) Sense: a combination of 2′F-RNA and 2′F-ANA in a 3-3 altimer design
- Antisense: 2′F-RNA only;
- (i) Sense: a combination of 2′F-RNA and 2′F-ANA in 3-3 and 1-1 altimer designs
- Antisense: 2′F-RNA only;
- (j) Sense: a combination of 2′F-RNA and 2′F-ANA in a 1-1 altimer design
- Antisense: 2′F-RNA only;
- (k) Sense: 2′F-ANA only
- Antisense: a combination of RNA and LNA;
- (l) Sense: a combination of 2′F-ANA and RNA
- Antisense: a combination of RNA and LNA;
- (m) Sense: a combination of 2′F-ANA, RNA and LNA
- Antisense: RNA only;
- (n) Sense: a combination of 2′F-ANA, RNA and LNA
- Antisense: a combination of RNA and LNA; or
- (o) Sense: a combination of 2′F-ANA, RNA and LNA
- Antisense: 2′F-RNA only.
- In a further embodiment, in the case of a sense strand comprising a 19 residue core (with or without an additional overhang), the sense strand comprises LNA residues at
positions - In a further embodiment, the above-mentioned oligonucleotide pair or duplex comprises:
-
(a) Sense: (2′F-RNA pyrimidines)x (2′F-ANA purines)y Antisense: (RNA)z wherein x is the number of pyrimidines and y is the number of purines in the sense strand, and wherein x + y = z. In an embodiment z = 19. (b) Sense: [(2′F-ANA)(2′F-RNA)](2′F-ANA) Antisense: (RNA)19 (c) Sense: (2′F-ANA)19 Antisense: (2′F-RNA)19 (d) Sense: [(2′F-ANA)3(2′F-RNA)3](2′F-ANA) Antisense: (RNA)19 (e) Sense: [(2′F-ANA)3(2′F-RNA)3][(2′F-ANA)(2′F-RNA)′(2′F-ANA) Antisense: (RNA)19 (f) Sense: (2′F-ANA)14 (RNA)5 Antisense: (2′F-RNA)19 (g) Sense: [(2′F-ANA)3(2′F-RNA)3](2′F-ANA) Antisense: (2′F-RNA)19 (h) Sense: [(2′F-ANA)3(2′F-RNA)3][(2′F-ANA)(2′F-RNA)]3(2′F-ANA) Antisense: (2′F-RNA)19 (i) Sense: [(2′F-ANA)(2′F-RNA)](2′F-ANA) Antisense: (2′F-RNA)19 (j) Sense: (2′F-ANA)19 Antisense: (LNA)(RNA)18 (k) Sense: (2′F-ANA)19 Antisense: (LNA)(RNA)(LNA)(RNA)16 (l) Sense: (2′F-ANA)10 Antisense: (RNA)11(LNA)2(RNA)6 (m) Sense: (2′F-ANA)(RNA)(LNA)(RNA)(2′F-ANA)4(RNA)2(LNA)(RNA)2(2′F- ANA)2(LNA)(RNA)(2′F-ANA)2 Antisense: (RNA)19 (n) Sense: (2′F-ANA)(RNARLNARRNA)(2′F-ANA)4(RNA)2(LNA)(RNA)2(2′F- ANA)(RNA)(2′F-ANA)(LNA)(2′F-ANA)(RNA) Antisense: (RNA)19 (o) Sense: (2′F-ANA)(RNA)(LNA)(RNA)(2′F-ANA)4(RNA)2(LNA)(RNA)2(2′F- ANA)(RNA)5 Antisense: (RNA)19 (p) Sense: (2′F-ANA)(RNA)(LNA)(RNA)(2′F-ANA)4(RNA)2(LNA)(RNA)2(2′F- ANA)2(LNA)(RNA)(2′F-ANA)2 Antisense: (LNA)(RNA)18 (q) Sense: (2′F-ANA)(RNA)(LNA)(RNA)(2′F-ANA)4(RNA)2(LNA)(RNA)2(2′F- ANA)(RNA)(2′F-ANA)(LNA)(2′F-ANA)(RNA) Antisense: (LNA)(RNA)18 (r) Sense: (2′F-ANA)(RNA)(LNA)(RNA)(2′F-ANA)4(RNA)2(LNA)(RNA)2(2′F- ANA)2(RNA)2(2′F-ANA)2 Antisense: (LNA)(RNA)18 (s) Sense: (2′F-ANA)(RNA)(LNA)(RNA)(2′F-ANA)4(RNA)2(LNA)(RNA)2(2′F- ANA)(RNA)(2′F-ANA)(RNA)(2′F-ANA)(RNA) Antisense: (LNA)(RNA)18 (t) Sense: (2′F-ANA)(RNA)(LNA)(RNA)(2′F-ANA)4(RNA)2(LNA)(RNA)2(2′F- ANA)(RNA)5 Antisense: (LNA)(RNA)18 (u) Sense: (2′F-ANA)(RNA)(LNA)(RNA)(2′F-ANA)4(RNA)2(LNA)(RNA)2(2′F- ANA)2(LNA)(RNA)(2′F-ANA)2 Antisense: (2′F-RNA)19 (v) Sense: (2′F-ANA)(RNA)(LNA)(RNA)(2′F-ANA)4(RNA)2(LNA)(RNA)2(2′F- ANA)(RNA)(2′F-ANA)(LNA)(2′F-ANA)(RNA) Antisense: (2′F-RNA)19 (w) Sense: (2′F-ANA)(RNA)(LNA)(RNA)(2′F-ANA)4(RNA)2(LNA)(RNA)2(2′F- ANA)2(RNA)2(2′F-ANA)2 Antisense: (2′F-RNA)19 (x) Sense: (2′F-ANA)(RNA)(LNA)(RNA)(2′F-ANA)4(RNA)2(LNA)(RNA)2(2′F- ANA)(RNA)(2′F-ANA)(RNA)(2′F-ANA)(RNA) Antisense: (2′F-RNA)19 or (y) Sense: (2′F-ANA)(RNA)(LNA)(RNA)(2′F-ANA)4(RNA)2(LNA)(RNA)2(2′F- ANA)(RNA)5 Antisense: (2′F-RNA)19. - In an embodiment, the above-mentioned oligonucleotide duplex comprises an overhang (e.g., a 5′ and/or 3′ overhang, on one strand or on both strands). In a further embodiment, the above-mentioned overhang is a 1 to 5 residues (e.g., nucleotides or modified nucleotides) overhang. In a further embodiment, the above-mentioned overhang is a 2 residues (e.g., nucleotides or modified nucleotides) overhang. For example, a 19 residue sense and/or antisense strand may comprise an overhang of an additional 1 to 5 residues. In such an example, a 2 residue overhang in both strands would result in sense and antisense strands of 21 residues each, 19 of which participate in base-pairing to form the duplex (the remaining 2 residues in each case representing the overhangs).
- In another embodiment, the above-mentioned overhang comprises DNA, 2′F-ANA and/or 2′F-RNA residues. In a further embodiment, the above-mentioned overhang comprises two 2′F-ANA residues. In a further embodiment, the above-mentioned overhang comprising two 2′F-ANA residues is on the sense strand.
- In another embodiment, the above-mentioned overhang comprises two 2′F-RNA residues. In a further embodiment, the above-mentioned overhang comprising two 2′F-RNA residues is on the antisense strand.
- In another embodiment, the above-mentioned overhang is a 3′ overhang.
- In another embodiment, the above-mentioned oligonucleotide pair or duplex is 5′ phosphorylated on one or both strands. In a further embodiment, the above-mentioned oligonucleotide pair or duplex is 5′ phosphorylated on the antisense strand.
- In embodiments, the sequence (e.g., nucleobase) complementarity between the sense strand and the antisense strand, or the sequence (e.g., nucleobase) identity between the sense strand and a target nucleic acid (e.g., mRNA), or a portion thereof, may be “perfect” or “complete” (100% complementarity or identity).
- In embodiments, the complementarity between the sense strand and the antisense strand, or the identity between the sense strand and a target nucleic acid (e.g., mRNA), or a portion thereof), is substantial, for example greater than about 70%. For example, for a duplex region consisting of 19 base pairs, one mismatch results in 94.7% complementarity, two mismatches results in about 89.5% complementarily, 3 mismatches results in about 84.2% complementarity, 4 mismatches results in about 79% complementarity and 5 mismatches results in about 74% complementarity. Accordingly, as used herein, “complementary” refers to both perfect complementarity and substantial complementarity between two sequences, for example to complementarity of greater than about 70% between the sequences. In an embodiment, the sense strand has an identity of at least 12 nucleotides, in a further embodiment of at least 12 contiguous nucleotides, to at least a portion of a target nucleic acid (e.g., mRNA). In an embodiment, the sense strand has an identity of at least 13 nucleotides, in further embodiments of at least 14, 15, 16, 17 or 18 nucleotides (contiguous or not), to at least a portion of a target nucleic acid. In another embodiment, the sense strand has complete identity to a portion of a target mRNA, with the exception of overhanging nucleotides (3′ overhang).
- Also, complementarity and identity as used herein refers to complementarity and identity of the nucleobase moieties (e.g., A, C, G, T or U), commonly referred to as “base pairing”, and is independent of for example modifications of the sugar moiety, such as those described herein. For example, a guanine nucleoside residue having any sugar moiety (i.e., modified or not) may base pair with a cytosine nucleoside residue similarly having any sugar moiety.
- “Identity” refers to sequence similarity between two peptides or two nucleic acid molecules. Identity can be determined by comparing each position in the aligned sequences. A degree of identity between nucleic acid or between amino acid sequences is a function of the number of identical or matching nucleotides or amino acids at positions shared by the sequences. As the term is used herein, a nucleic acid sequence is “substantially identical” to another sequence if the functional activity of the sequences is conserved. Two nucleic acid sequences are considered substantially identical if, when optimally aligned (with gaps permitted), they share at least about 70% sequence similarity or identity, or if the sequences share defined functional motifs. In alternative embodiments, sequence similarity in optimally aligned substantially identical sequences may be at least 75%, 80%, 85%, 90% or 95%. An “unrelated” sequence shares less than 40% identity, though preferably less than about 25% identity, with a given reference sequence (e.g., a target nucleic acid).
- Substantially complementary nucleic acids are nucleic acids in which the complement of one molecule is substantially identical to the other molecule. Two nucleic acid or protein sequences are considered substantially identical if, when optimally aligned, they share at least about 70% sequence identity. In alternative embodiments, sequence identity may for example be at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. Optimal alignment of sequences for comparisons of identity may be conducted using a variety of algorithms, such as the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math 2: 482, the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443, the search for similarity method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85: 2444, and the computerised implementations of these algorithms (such as GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, Wis., U.S.A.). Sequence identity may also be determined using the BLAST algorithm, described in Altschul et al., 1990, J. Mol. Biol. 215: 403-10 (using the published default settings). Software for performing BLAST analysis may be available through the National Center for Biotechnology Information. The BLAST algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold. Initial neighbourhood word hits act as seeds for initiating searches to find longer HSPs. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction is halted when the following parameters are met: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program may use as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (Henikoff and Henikoff, 1992, Proc. Natl. Acad. Sci. USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10 (or 1 or 0.1 or 0.01 or 0.001 or 0.0001), M=5, N=4, and a comparison of both strands. One measure of the statistical similarity between two sequences using the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. In alternative embodiments of the invention, nucleotide or amino acid sequences are considered substantially identical if the smallest sum probability in a comparison of the test sequences is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
- An alternative indication that two nucleic acid sequences are substantially complementary is that the two sequences hybridize to each other under moderately stringent, or preferably stringent, conditions. Hybridization to filter-bound sequences under moderately stringent conditions may, for example, be performed in 0.5 M NaHPO4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.2×SSC/0.1% SDS at 42° C. (see Ausubel, et al. (eds), 1989, Current Protocols in Molecular Biology, Vol. 1, Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York, at p. 2.10.3). Alternatively, hybridization to filter-bound sequences under stringent conditions may, for example, be performed in 0.5 M NaHPO4, 7% SDS, 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at 68° C. (see Ausubel, et al. (eds), 1989, supra). Hybridization conditions may be modified in accordance with known methods depending on the sequence of interest (see Tijssen, 1993, Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, Part I,
Chapter 2 “Overview of principles of hybridization and the strategy of nucleic acid probe assays”, Elsevier, New York). Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point for the specific sequence at a defined ionic strength and pH. - The sense strand and the antisense strand may be linked by a loop structure, which may be comprised of a non-nucleic acid polymer such as, inter alia, polyethylene glycol. Alternatively, the loop structure may be comprised of a nucleic acid, including modified and non-modified ribonucleotides and modified and non-modified deoxyribonucleotides.
- In an embodiments, the 5′-terminus of the sense strand of the oligonucleotide duplex may be linked to the 3′-terminus of the antisense strand, or the 3 ‘-terminus of the sense strand may be linked to the 5 ’-terminus of the sense strand, said linkage being via a nucleic acid linker typically having a length between 2 to 100 nucleotides (or modified nucleotides), preferably about 2 to about 30 nucleobases.
- In an embodiment, the above-mentioned oligonucleotide duplex is a hairpin duplex, that is a single strand comprising the sense and antisense strands which is self-complementary and folds back onto itself.
- The invention further provides a salt, such as a pharmaceutically acceptable salt, of any of the above-mentioned compounds (e.g., oligonucleotide, oligonucleotide duplex, siRNA or siRNA-like molecule) where applicable.
- The present invention also relates to compounds which down-regulate expression of various genes, i.e., decrease production of an encoded polypeptide. The invention provides oligonucleotides/oligonucleotide duplexes of the invention and uses thereof in siRNA/RNAi applications, whereby expression of a nucleic acid encoding a polypeptide of interest, or a fragment thereof, may be inhibited or prevented using RNA interference (RNAi) technology, a type of post-transcriptional gene silencing. RNAi may be used to create a pseudo “knockout”, i.e., a system in which the expression of the product encoded by a gene or coding region of interest is reduced, resulting in an overall reduction of the activity of the encoded product in a system. As such, RNAi may be performed to target a nucleic acid of interest or fragment or variant thereof, to in turn reduce its expression and the level of activity of the product which it encodes. Such a system may be used for functional studies of the product, as well as to treat disorders related to the activity of such a product. RNAi is described in for example U.S. patent publications Nos. 2002/0173478 (Gewirtz; published Nov. 21, 2002) and 2002/0132788 (Lewis et al.; published Nov. 7, 2002). Reagents and kits for performing RNAi are available commercially from for example Ambion Inc. (Austin, Tex., USA), New England Biolabs Inc. (Beverly, Mass., USA) and Invitrogen (Carlsbad, Calif., USA).
- The initial agent for RNAi in some systems is thought to be dsRNA or modified dsRNA molecules corresponding to a target nucleic acid. The dsRNA is then thought to be cleaved into short interfering RNAs (siRNAs) which are for example 21-23 nucleotides in length (19-21 by duplexes, each with 2
nucleotide 3′ overhangs). The enzyme thought to effect this first cleavage step (the Drosophila version is referred to as “Dicer”) is categorized as a member of the RNase III family of dsRNA-specific ribonucleases. Alternatively, RNAi may be effected via directly introducing into the cell, or generating within the cell by introducing into the cell an siRNA or siRNA-like molecule or a suitable precursor (e.g., vector encoding precursor(s), etc.) thereof. An siRNA may then associate with other intracellular components to form an RNA-induced silencing complex (RISC). The RISC thus formed may subsequently target a transcript of interest via base-pairing interactions between its siRNA component and the target transcript by virtue of homology, resulting in the cleavage of the target transcript approximately 12 nucleotides from the 3′ end of the siRNA. Thus the target mRNA is cleaved and the level of protein product it encodes is reduced. - RNAi may be effected by the introduction of suitable in vitro synthesized siRNA or siRNA-like molecules into cells. RNAi may for example be performed using chemically-synthesized RNA or modified RNA molecules. Alternatively, suitable expression vectors may be used to transcribe such RNA either in vitro or in vivo. In vitro transcription of sense and antisense strands (encoded by sequences present on the same vector or on separate vectors) may be effected using for example T7 RNA polymerase, in which case the vector may comprise a suitable coding sequence operably-linked to a T7 promoter. The in vitro-transcribed RNA may in embodiments be processed (e.g., using E. coli RNase III) in vitro to a size conducive to RNAi. The sense and antisense transcripts are combined to form an RNA duplex which is introduced into a target cell of interest. Other vectors may be used, which express small hairpin RNAs (shRNAs) which can be processed into siRNA-like molecules. Various vector-based methods have been described (see, e.g., Brummelkamp et al. [2002] Science 296: 550). Various methods for introducing such vectors into cells, either in vitro or in vivo (e.g. gene therapy) are known in the art.
- Accordingly, in an embodiment of the invention, a nucleic acid, either a non-coding RNA (ncRNA) as well as an RNA encoding a polypeptide of interest (e.g. an mRNA), or a fragment thereof, may be inhibited by introducing into or generating within a cell an siRNA or siRNA-like molecule based on an oligonucleotide of the invention, corresponding to a nucleic acid of interest, or a fragment thereof, or to an nucleic acid homologous thereto (sometimes collectively referred to herein as a “target nucleic acid”). “Target nucleic acid” as used herein refers to a nucleic acid encoding a polypeptide (e.g., a coding RNA such as a mRNA), as well as to a non-coding nucleic acid, such as a non-coding RNA (ncRNA), i.e., an RNA that is not translated to a protein and which are involved in various cell functions including post-transcriptional modifications, gene regulation and propagation (virus). Examples of ncRNA include transfer RNA (tRNA), ribosomal RNA (rRNA) and small nuclear RNA (snRNA). As such, degradation and a decrease in level of the target nucleic acid may be effected, and in the case of a target nucleic which encodes a polypeptide, a decrease in the production or level of the polypeptide may be effected.
- “siRNA-like molecule” refers to a nucleic acid molecule similar to an siRNA (e.g., in size and structure) and capable of eliciting siRNA activity, i.e., to effect the RNAi-mediated inhibition of production of the polypeptide. In various embodiments such a method may entail the direct administration of the siRNA or siRNA-like molecule into a cell. In an embodiment, the siRNA or siRNA-like molecule is less than about 30 nucleotides in length. In a further embodiment, the siRNA or siRNA-like molecule is about 19-23 nucleotides in length. In an embodiment, siRNA or siRNA-like molecule comprises a 19-21 by duplex portion, each strand having a 2
nucleotide 3′ overhang. In other embodiments, one or both strands may have blunt ends. In embodiments, the siRNA or siRNA-like molecule is substantially identical to a nucleic acid encoding a polypeptide of interest, or a fragment or variant (or a fragment of a variant) thereof. Such a variant is capable of encoding a protein having activity similar to the polypeptide of interest. - Accordingly, the present invention further provides a double-stranded siRNA or siRNA-like molecule (or modified siRNA) comprising an oligonucleotide duplex of the invention.
- It is to be understood that, in the context of the present invention, any of the oligonucleotide duplexes or siRNA/siRNA-like molecules disclosed herein, or any long double-stranded RNA molecules (typically 25-500 nucleotides in length) which are processed by endogenous cellular complexes (such as Dicer or a counterpart thereof—see above) to form the siRNA molecules disclosed herein, or molecules which comprise the oligonucleotide duplexes or siRNA molecules disclosed herein, are within the scope of the present invention. For example, it is envisaged that a long oligonucleotide (e.g., of about 80 to 500 nucleotides in length) comprising one or more stem and loop structures, where stem regions comprise the oligonucleotides of the invention, may be delivered in a carrier, preferably a pharmaceutically acceptable carrier, and may be processed intracellularly by endogenous cellular complexes to produce one or more smaller double stranded oligonucleotides (siRNA/siRNA-like molecules) of the present invention. This oligonucleotide is typically referred to as a tandem shRNA construct.
- In an embodiment, the above-mentioned siRNA is a 25 to 30 nucleotides, which may be substrates for the Dicer endonuclease (Kim D.-M. et al. Nature Biotechnology, vol. 23, pp. 222-226 (2005)).
- The present invention also provides a composition (e.g., a pharmaceutical composition) comprising an oligonucleotide, oligonucleotide duplex or siRNA-like molecule of the invention, and an excipient or carrier, such as a biologically or pharmaceutically acceptable carrier or excipient. In one embodiment, such compositions include an oligonucleotide, oligonucleotide duplex or siRNA-like molecule of the invention in a therapeutically or prophylactically effective amount sufficient to treat a condition/disease associated with the expression (e.g., overexpression) of a target nucleic acid, and/or of a polypeptide encoded by a target nucleic acid. The therapeutic composition may be soluble in an aqueous solution at a physiologically acceptable pH.
- As used herein “pharmaceutically acceptable carrier” or “excipient” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are physiologically compatible. In one embodiment, the carrier is suitable for parenteral administration. Alternatively, the carrier can be suitable for intravenous, intraperitoneal, intramuscular, topical, sublingual or oral administration, or for administration by inhalation. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.
- Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin. Moreover, an oligonucleotide of the invention can be administered in a time release formulation, for example in a composition which includes a slow release polymer. The active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers (PLG). Many methods for the preparation of such formulations are patented or generally known to those skilled in the art.
- Sterile injectable solutions can be prepared by incorporating the active compound (e.g. an oligonucleotide, oligonucleotide duplex, siRNA, or siRNA-like molecule of the invention) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. In accordance with an alternative aspect of the invention, an oligonucleotide of the invention may be formulated with one or more additional compounds that enhance its solubility.
- Suitable methods for siRNA delivery to effect RNAi according to embodiments include any method by which a siRNA can be introduced into an organelle, a cell, a tissue or an organism, as described herein or as would be known to one of ordinary skill in the art. Such methods include, but are not limited to, direct delivery of siRNA such as by injection including microinjection, electroporation, calcium phosphate precipitation, using DEAE-dextran followed by polyethylene glycol, direct sonic loading, liposome-mediated transfection, microprojectile bombardment, agitation with silicon carbide fibers, Agrobacterium-mediated transformation, PEG-mediated transformation, desiccation/inhibition-mediated uptake, and the like. Through the use of techniques such as these, an organelle, cell, tissue or organism may be stably or transiently transformed. The oligonucleotide, double stranded molecule/duplex, siRNA molecule or composition of the invention may be delivered in liposome or lipofectin formulations and the like and are prepared by methods well known to those skilled in the art. Such methods are described, for example, in U.S. Pat. Nos. 5,593,972, 5,589,466, and 5,580,859.
- Delivery systems aimed specifically at the enhanced and improved delivery of siRNA into mammalian cells have been developed (see, for example, Shen et al. FEBS Let. 2003, 539: 111-114; Xia et al., Nat. Biotech. 2002, 20: 1006-1010; Sorensen et al., J. Mol. Biol. 2003. 327: 761-766; Lewis et al., Nat. Gen. 2002, 32: 107-108 and Simeoni et al., Nucleic Acids Research 2003, 31(11): 2717-2724).
- In accordance with another aspect of the invention, therapeutic compositions of the present invention, comprising an oligonucleotide duplex, siRNA, or siRNA-like molecule of the invention, may be provided in a kit or commercial package. The kit may further comprise instructions for the use of the oligonucleotide duplex, siRNA, or siRNA-like molecule for the inhibition of a target gene expression, and/or prevention and/or treatment of a disease/condition associated with expression (e.g., overexpression) of a target nucleic acid or gene. The kit may further comprise a validated positive control siRNA that targets a housekeeping gene and/or a validated negative control siRNA that is nontargeting. The kit may further comprise one or more reagents, such as reagents for introducing the oligonucleotide duplex, siRNA, or siRNA-like molecule of the invention into a cell (e.g., transfection/transformation reagents) and/or reagents for assessing knockdown of the intended target gene such as antibodies for monitoring knockdown at the protein level by immunofluorescence or Western analysis, reagents for assessing enzymatic activity or presence of a reporter protein, or reagents for assessing cell viability. RT-PCR primers and probes may be included for detection of target or reporter mRNA. The kit may further comprise a container (e.g., vial, test tube, flask, bottle, syringe or other packaging means) into which the oligonucleotide duplex, siRNA, or siRNA-like molecule may be placed/aliquoted, as well as devices for administering the oligonucleotide duplex, siRNA, or siRNA-like molecule to a subject (e.g., syringe).
- The invention further provides a method of inhibiting the expression of a target gene/nucleic acid, or of degrading or decreasing the level of a target gene/nucleic acid, in a biological system (e.g., a cell, a tissue, an organ, a subject), e.g., to inhibit production of a polypeptide encoded by the target gene/nucleic acid, comprising introducing into the system the above-mentioned oligonucleotide duplex, siRNA or siRNA-like molecule.
- According to another aspect of the invention, a method of inhibiting production of the product of a gene (“gene silencing”; e.g., of a deleterious gene) in a patient in need thereof is provided. “Gene silencing” as used herein refers to an inhibition or reduction of the expression of the protein encoded by a particular nucleic acid sequence or gene (e.g., a deleterious gene). The method comprises administering to the patient a therapeutically effective amount of oligonucleotide, a double stranded molecule/duplex, an siRNA molecule or a composition of the invention. In embodiments, the target gene or nucleic acid is a viral, bacterial or mammalian (e.g., human) gene.
- The invention further provides a method of treating a condition associated with expression of a gene/nucleic acid in a subject, e.g., associated with the production of a polypeptide encoded by the target gene/nucleic acid, the method comprising administering the oligonucleotide duplex, siRNA or siRNA-like molecule to the subject (or to a cell, tissue, organ from the subject), wherein the siRNA or siRNA-like molecule is targeted to (or specific for) the gene/nucleic acid.
- The invention further provides a use of the siRNA or siRNA-like molecule for the preparation of a medicament.
- The invention further provides a use of the above-mentioned siRNA or siRNA-like molecule for a method selected from: (a) gene silencing; (b) inhibiting gene expression/polypeptide production in a biological system; (c) inhibiting gene expression/polypeptide production in a subject; (d) degrading or decreasing the level of a target gene/nucleic acid in a biological system or a subject; (d) treating a disease/condition associated with the production of a polypeptide encoded by a gene/nucleic acid in a subject; and (e) preparation of a medicament, for example a medicament for treating a disease or condition associated with expression (e.g., overexpression) of a nucleic acid/gene in a subject.
- In various embodiments, an oligonucleotide pair, duplex, siRNA and/or siRNA-like molecule of the invention may be used prophylactically and/or therapeutically in formulations or medicaments to prevent or treat a disease/condition associated with the expression of a target nucleic acid or gene. The invention provides corresponding methods of medical treatment, in which a therapeutic dose of an oligonucleotide of the invention is administered in a pharmacologically acceptable formulation, e.g., to a patient or subject in need thereof.
- A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as a reduction or reversal in progression of a disease associated with the production of a polypeptide encoded by a target nucleic acid or gene. A therapeutically effective amount of an oligonucleotide pair, duplex, siRNA and/or siRNA-like molecule of the invention may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental effects of the compound are outweighed by the therapeutically beneficial effects. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as preventing or inhibiting the rate of onset or progression of a disease associated with the production of a polypeptide encoded by a target nucleic acid or gene. A prophylactically effective amount can be determined as described above for the therapeutically effective amount. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgement of the person administering or supervising the administration of the compositions.
- The invention further provides a use of an oligonucleotide, pair or duplex of the invention or the above-mentioned composition for degrading or decreasing the level of a target nucleic acid, or of decreasing the production or the level of a polypeptide encoded by a target nucleic acid or gene or for the prevention and/or treatment of a disease/condition associated with production of a polypeptide encoded by a target nucleic acid or gene. The invention further provides a use of an oligonucleotide of the invention for the preparation of a medicament. In an embodiment, the medicament is for prevention and/or treatment of a disease or condition associated with expression (e.g., overexpression) of a target nucleic acid or gene.
- The target gene/nucleic acid can be a gene/nucleic acid derived from a cell, an endogenous gene, a transgene, or exogenous genes such as genes of a pathogen, for example, a virus, which is present in the cell after infection thereof. The cell having the target gene may be from the germ line or somatic, totipotent or pluripotent, dividing or non-dividing, parenchyma or epithelium, immortalized or transformed, or the like. The cell can be a gamete or an embryo; if an embryo, it can be a single cell embryo or a constituent cell or cells from a multicellular embryo. The term “embryo” thus encompasses fetal tissue. The cell having the target gene may be an undifferentiated cell, such as a stem cell, or a differentiated cell, such as from a cell of an organ or tissue, including fetal tissue, or any other cell present in an organism. Cell types that are differentiated include adipocytes, fibroblasts, myocytes, cardiomyocytes, endothelium, neurons, glia, blood cells, megakaryocytes, lymphocytes, macrophages, neutrophils, eosinophils, basophils, mast cells, leukocytes, granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts, hepatocytes, and cells, of the endocrine or exocrine glands.
- The oligonucleotide pair, duplex, siRNA or siRNA-like molecule of the invention may be associated with, for example, a cell-targeting ligand. As used herein, a “cell targeting ligand” is a cell-directing molecule that has specificity for targeted sites such as cell surface receptors. This allows, for example, a more specific delivery of the oligonucleotide pair, duplex, siRNA or siRNA-like molecule to a particular cell/cell type, tissue or organ.
- In another aspect, the present invention provides a method for increasing/improving the efficacy, potency and/or stability (e.g., in vivo stability) of an oligonucleotide duplex, comprising incorporating into said duplex (a) one or more 2′-substituted arabinonucleotides (ANA); and (b) (i) one or more 2′-substituted ribonucleotides (RNA), (ii) one or more locked nucleic acid nucleotides (LNA), or (iii) a combination of (i) and (ii).
- In another aspect, the present invention provides a method for reducing off-target effects of an oligonucleotide duplex, comprising incorporating into said duplex (a) one or more 2′-substituted arabinonucleotides (ANA); and (b) (i) one or more 2′-substituted ribonucleotides (RNA), (ii) one or more locked nucleic acid nucleotides (LNA), or (iii) a combination of (i) and (ii).
- The invention further provides a method of synthesizing an oligonucleotide of the invention, the method comprising: (a) 5′-deblocking; (b) coupling; (c) capping; and (d) oxidation; wherein (a), (b), (c) and (d) are repeated under conditions suitable for the synthesis of the oligonucleotide, wherein the synthesis is carried out in the presence of a suitable nucleotide monomer described herein (e.g., RNA, DNA, 2′F-ANA, 2′F-RNA, LNA).
- The invention further provides a method to prepare an oligonucleotide duplex of the invention comprising combining a first (e.g., sense) strand comprising an oligonucleotide of the invention and a second (e.g., antisense) strand substantially complementary to the first strand under conditions permitting formation of a duplex via base-pairing between the first and second strands.
- In embodiments, the synthesis is carried out on a solid phase, such as on a solid support selected from the group consisting of controlled pore glass, polystyrene, polyethylene glycol, polyvinyl, silica gel, silicon-based chips, cellulose paper, polyamide/kieselgur and polacryloylmorpholide. In further embodiments, the monomers may be used for solution phase synthesis or ionic-liquid based synthesis of oligonucleotides.
- “5′-Deblocking” as used herein refers to a step in oligonucleotide synthesis wherein a protecting group is removed from a previously added nucleoside (or a chemical group linked to a solid support), to produce a reactive hydroxyl which is capable of reacting with a nucleoside molecule, such as a nucleoside phosphoramidite or H-phosphonate.
- “Protecting group” as used herein refers to a moiety that is temporarily attached to a reactive chemical group to prevent the synthesis of undesired products during one or more stages of synthesis. Such a protecting group may then be removed to allow for step of the desired synthesis to proceed, or to generate the desired synthetic product. Examples of protecting groups are trityl (e.g., monomethoxytrityl, dimethoxytrityl), silyl, levulinyl and acetyl groups.
- “Coupling” as used herein refers to a step in oligonucleotide synthesis wherein a nucleoside is covalently attached to the terminal nucleoside residue of the oligonucleotide (or to the solid support via for example a suitable linker), for example via nucleophilic attack of an activated nucleoside phosphoramidite, H-phosphonate, phosphotriester, pyrophosphate, or phosphate in solution by a
terminal 5′-hydroxyl group of a nucleotide or oligonucleotide bound to a support. Such activation may be effected by an activating reagent such as tetrazole, 5-ethylthio-tetrazole, 4,5-dicyanoimidazole (DCI), and/or pivaloyl chloride. - “Capping” as used herein refers to a step in oligonucleotide synthesis wherein a chemical moiety is covalently attached to any free or unreacted hydroxyl groups on the support bound nucleic acid or oligonucleotide (or on a chemical linker attached to the support). Such capping is used to prevent the formation of for example sequences of shorter length than the desired sequence (e.g., containing deletions). An example of a reagent which may be used for such capping is acetic anhydride. Further, the capping step may be performed either before or after the oxidation (see below) of the phosphite bond.
- “Oxidation” as used herein refers to a step in oligonucleotide synthesis wherein the newly synthesized phosphite triester or H-phosphonate diester bond is converted into pentavalent phosphate triester or diester bond. In the case where a phosphorothioate internucleotide linkage is desired, “oxidation” also refers to the addition of a sulfur atom to generate a phosphorothioate linkage.
- The following examples are illustrative of various aspects of the invention, and do not limit the broad aspects of the invention as disclosed herein.
- The present invention is illustrated in further details by the following non-limiting examples.
- Oligonucleotide synthesis. Standard conditions for solid-phase oligonucleotide synthesis were used for the synthesis of all oligonucleotides, at a 0.8 to 1.0 μmol scale. 4,5-Dicyanoimidazole (0.50 M in acetonitrile) or 5-ethylthiotetrazole (0.25 M in acetonitrile) were used as activators, and 0.10 M iodine in 1:2:10 pyridine:water:THF was used as oxidant (wait time during the oxidation step was 24 seconds). Phosphoramidites were prepared as 0.15 M solutions (RNA amidites) or 0.08-0.10 M solutions (DNA, 2′-fluoro amidites). Coupling times were extended to 10-30 minutes for modified nucleotides. The oligonucleotides were treated with 3:1 ammonium hydroxide:ethanol for 16 h at 55° C. to cleave them from the solid support and deprotect the phosphates and bases. Sequences containing ribonucleotides were concentrated and desilylated with Et3N.3HF (100 μL) for 48 h at room temperature. Sequence purification was accomplished by anion exchange HPLC using 0-0.2 M LiClO4 solution as eluent, or by preparative denaturing PAGE. Desalting was effected on Sephadex G-25 or NAP-25 columns. Sequence purity was verified using denaturing PAGE.
- 5′-phosphorylation of oligonucleotides was generally accomplished on the CPG solid support, by treating the newly-synthesized oligonucleotide with bis(2-cyanoethyl)-diisopropylaminophosphoramidite and ethylthiotetrazole, followed by normal deprotection conditions. ESI-MS was used to confirm the success of the phosphorylation reaction.
- Thermal denaturation and CD studies. Equimolar amounts of complementary sequences were combined, dried and rediluted in pH 7.2 buffer containing 140 mM KCl, 1 mM MgCl2 and 5 mM NaHPO4 (1 mL). After heating to 90° C., the samples were slowly cooled to room temperature and refrigerated overnight. They were then transferred into cold cuvettes in a Cary™ 300 UV spectrophotometer. The change in absorbance at 260 nm was then monitored upon heating from 15° C. to 90° C. Melting temperatures were determined as the maxima of the first derivatives or using the baseline method, as implemented in the Varian™ software.
- CD spectra were obtained on a Jasco™ J-720 spectropolarimeter at 20° C. using samples annealed in the same buffer and under the same conditions as for the thermal denaturation studies. Spectra were baseline-corrected with respect to a blank containing the buffer but no duplex. Smoothing and adjustment for duplex concentration were effected using the Spectra-Manager program (Jasco).
- siRNA assays (luciferase inhibition). HeLa X1/5 cells that stably express firefly luciferase were grown as previously described (Wu, H. et al. J. Biol. Chem. 1999, 274: 28270-28278). The day prior to transfection, 0.5×105 cells were plated in each well of a 24-well plate. The next day, the cells were incubated with increasing amounts of siRNAs premixed with lipofectamine-plus™ reagent (Invitrogen) using 1 μL of lipofectamine and 4 μL of the plus reagent per 20 pmol of siRNA (for the highest concentration tested). For the siRNA titrations, each siRNA was diluted into dilution buffer (30 mM HEPES-KOH, pH 7.4, 100 mM KOAc, 2 mM MgOAc2) and the amount of lipofectamine-plus reagent used relative to the siRNAs remained constant. 24 hours after transfection, the cells were lysed in hypotonic lysis buffer (15 mM K3PO4, 1 mM EDTA, 1% Triton, 2 mM NaF, 1 mg/ml BSA, 1 mM DTT, 100 mM NaCl, 4 μg/mL aprotinin, 2 μg/mL leupeptin and 2 μg/mL pepstatin) and the firefly light units were determined using a Fluostar Optima 96-well plate bioluminescence reader (BMG Labtech) using firefly substrate as described (Novac, O. et al. J. Nucl. Acids Res. 2004, 32: 902-915). The luciferase counts were normalized to the protein concentration of the cell lysate as determined by the DC protein assay (BioRad). Error bars represent the standard deviation of at least four transfections. Cotransfecting the siRNAs and the plasmid pCl-hRL-con expressing the Renilla luciferase mRNA (Pillai, R. S. et al. Science 2005, 309: 1573-1576) in the same cell line showed no difference in expression of this reporter, demonstrating the specificity of the RNAi effects.
- Assessment of IFN production using the HEK-Blue™ IFN detection assay. 48 hours after siRNA transfection, cells were left untreated or treated with 1 ug/ml of poly(I:C) for 24 hours. The amount of IFN in the supernatant was measured according to the manufacturer's instructions (InvivoGen). Briefly, supernatants were mixed with HEK-Blue™ cells that carry a reporter gene expressing a secreted alkaline phosphatase under the control of the interferon stimulated response element 9 (ISRE9) promoter. In response to IFN exposure, the HEK-Blue™ cells release soluble alkaline phosphatase that is quantified by mixing the supernatant with Quanti Blue™ (InvivoGen) reagent and measuring the absorbance at 650 nm.
- A series of duplexes containing fully-modified 2′F-ANA and 2′F-RNA strands were made (Table I). These duplexes target positions 1818-1836 of the firefly luciferase gene (RefSeq accession number M15077). A series of chimeric strands containing both 2′-fluoro epimers was also designed. One chimera consisted of 2′F-RNA pyrimidines and 2′F-ANA purines. Another pair of strands was a “1-1 altimer” structure, with alternating 2′F-ANA and 2′F-RNA residues. For all of these 2′F-ANA/2′F-RNA chimeric strands, the 3′-overhang was always made of 2′F-ANA.
-
TABLE I Sequences of the siRNAs targeting positions 1818-1836 of firefly luciferase containing mixtures of 2′F-ANA and 2′F-RNA. SEQ ID Name Description Sequence Tm NO: jg-1 RNA 5′-GCUUGAAGUCUUUAAUUAAtt-3′ 61.8 19 RNA 5′-UUAAUUAAAGACUUCAAGCgg-3′ 20 jg-2 pur/ pyr 5′- G GAAG AA AATT -3′ 65.6 21 pur/ pyr 5′- AA AAAGA AAG GG -3′ 22 jg-3 1-1 altimer 5′- G T G A T T T A T TT -3′ 36.8 23 1-1 altimer 5′- T A T A G C T C A G G G -3 24 jg-4 2′F- RNA 5′- -3′ >90 25 5′-p -3′ 26 jg-5 2′F- ANA 5′-GCTTGAAGTCTTTAATTAATT-3′ 72.8 27 5′-pTTAATTAAAGACTTCAAGCGG-3′ 28 jg-6 pur/ pyr 5′- G GAAG AA AATT -3′ 62.5 21 RNA 5′-UUAAUUAAAGACUUCAAGCgg-3′ 20 jg-7 RNA 5′-GCUUGAAGUCUUUAAUUAAtt-3′ 56.7 19 pur/ pyr 5′- AA AAAGA AAG GG -3′ 22 jg-8 1-1 altimer 5′- G T G A T T T A T ATT -3′ 48.2 23 RNA 5′-UUAAUUAAAGACUUCAAGCgg-3′ 20 jg-9 RNA 5′-GCUUGAAGUCUUUAAUUAAtt-3′ 45.8 19 1-1 altimer 5′- T A T A G C T A G GG -3 24 jg-10 2′F- RNA 5′- -3′ 76.5 25 RNA 5′-UUAAUUAAAGACUUCAAGCgg-3′ 20 jg-11 RNA 5′-GCUUGAAGUCUUUAAUUAAtt-3′ 76.2 19 2′F- RNA 5′-p -3′ 26 jg-12 2′F- ANA 5′- GCTTGAAGTCTTTAATTAATT -3′ 64.7 27 RNA 5′-UUAAUUAAAGACUUCAAGCgg-3′ 20 jg-13 RNA 5′-GCUUGAAGUCUUUAAUUAAtt-3′ 62.8 19 2′F- ANA 5′-p TTAATTAAAGACTTCAAGCGG -3′ 28 jg-14 2′F- ANA 5′- GCTTGAAGTCTTTAATTAATT -3′ 80.1 27 2′F- RNA 5′-p -3′ 26 jg-15 2′F- RNA 5′- -3′ 77.5 25 2′F- ANA 5′-p TTAATTAAAGACTTCAAGCGG -3′ 28 Uppercase = RNA Lowercase = dna Uppercase bold underline = 2 ′ F-ANA (FANA) Uppercase bold italic = 2′ p = 5-Phosphate - The RNAi activity of all duplexes was tested under the same conditions described above. Results are shown in
FIG. 1 . - Four of the duplexes (jg-6, jg-8, jg-10 and jg-12) contained a modified sense strand paired with an RNA antisense strand. The best of these four duplexes is jg-6, containing a purine/pyrimidine chimeric sense strand. The second-best duplex is duplex jg-8, containing the 1-1 altimer configuration in the sense strand. Thus, combining the two 2′-F epimers in the sense strand yields better results than using either chemistry alone, and strikingly, with better results relative to the natural RNA (jg-1).
- Comparison of the RNAi activity of duplexes jg-6-jg-13 allows to evaluate the appropriateness of each type of modified strand architecture (2′F-ANA, 2′F-RNA, purine/pyrimidine and 1-1 altimer) in the sense or antisense strands. Sense/antisense preferences are observed for all four types of modified strands. Duplexes jg-6, jg-8 and jg-12 are more active than jg-7, jg-9 and jg-13, respectively, revealing that both chimeric constructs and the 2′F-ANA strand are better-tolerated in the sense strand than the antisense strand. The difference is particularly striking between duplexes jg-8 and jg-9 containing one 1-1 altimer strand; jg-8 (1-1 altimer in the sense strand) was one of the most active duplexes tested, while jg-9 (1-1 altimer in the antisense strand) was inactive.
-
FIG. 1 shows that jg-11 is more active than jg-10, thus suggesting that 2′F-RNA is better-tolerated in the antisense than the sense strand. It is believed that this is the first time a fully-modified or heavily-modified strand has been observed to be better tolerated in the antisense than the sense strand. - A 2′F-ANA sense strand and a 2′F-RNA antisense strand formed a duplex that was found to be active as well. Indeed, synergy between these two modifications is observed in the case of duplex jg-14, which is more active than either of the duplexes jg-11 or jg-12 from which it is derived. On the other hand, reversing the sense/antisense combination gave jg-15, one of the least potent siRNAs tested in this study.
- The thermal stabilities of the duplexes were tested by heating the annealed duplexes, in physiological buffer, and measuring the change in the absorbance at 260 nm (A260). Binding affinities of the modified duplexes vary widely. There was no correlation between RNAi activity and binding affinity. For example, two of the most active duplexes we tested were jg-4 and jg-8, with Tm values of >90° C. and 48.2° C., respectively. The most potent duplex, the fully fluorinated heteroduplex jg-14, had a Tm about 20° C. higher than that that of native RNA duplex (80.1° C. vs 61.8° C.).
- The CD spectra of the modified duplexes were examined, to explore possible connections between helical structure and siRNA activity. Results are presented in
FIG. 2 . The changes in the Cotton effects at 210-220 nm are noteworthy. Beginning with duplexes jg-2-jg-5, which have the same chemistry in both strands, it is noteworthy that for 2′F-RNA duplex jg-4, this band is of maximum intensity at 227 nm, which is slightly redshifted with respect to the control duplex jg-1 (224 nm). On the other hand, for the three duplexes containing 2′F-ANA, including the two chimeric architectures jg-2 and jg-3 and the all-2′F-ANA duplex jg-5, this band is blueshifted and reaches maximum intensity at about 220 nm. Furthermore, duplexes jg-1 and jg-4 feature a more strongly negative band at 210 nm. This is consistent with the degree of A-form helicity of the duplexes (Ratmeyer, L. et al. Biochemistry 1994, 33: 5298-5304). 2′F-RNA duplex jg-4 also has the highest intensity for its 270 nm band, followed by native RNA duplex jg-1, then the 2F-ANA-containing strands. Fully-2′F-ANA duplex jg-5 is quite B-form in character, as evidenced by the fact that its 270 nm band is of the lowest intensity and contains a shoulder above 280 nm, and its 245 nm negative band is significantly more negative than the other duplexes (Ratmeyer, L. et al. 1994, supra). - For duplexes jg-6-jg-13, a modified sense strand corresponded to higher molar ellipticity at 220 nm than was observed for the native and antisense-modified duplexes. Thus, the intensity of the 220 nm band for the various sense antisense pairs jg-6/jg-7, jg-8/jg-9, jg-10/jg-11 and jg-12/jg-13 was always higher for the first member of each pair. Because sense modification led to higher potency for 3 of the 4 modified strand architectures, this higher intensity also corresponded with higher potency, with the exception of duplexes jg-10 and jg-11, for which the 2′F-RNA-modified strand was better-accepted in the antisense than the sense. It is also interesting that modifying the sense strand, but not the antisense strand, with 2′F-RNA, led to a notable increase in the intensity of the Cotton effects at 270 nm.
- For duplexes jg-14 and jg-15, in which both strands were modified, the more potent duplex jg-14 featured higher intensity for its 220 nm band, and indeed, in the whole range from 205-250 nm. It is not clear why such a large difference is observed between these two duplexes at lower wavelengths. Duplex jg-15 should have more A-form character since it has more strongly negative peaks at 210 nm, but the higher Tm of jg-14 implies that it has more A-form character than jg-15 (Ratmeyer, L. et al. 1994, supra).
- To investigate whether the potency and synergy obtained for 2′F-ANA-2′F-RNA combinations was applicable to other siRNA sequences, other duplexes directed against the same gene and cell line, this time targeting positions 515-533 (Hoshika, S. et al. FEBS Lett. 2005, 579: 3115-3118; Elbashir, S. M. et al. Nature 2001, 411: 494-498). A series of fully or heavily 2′-fluorinated duplexes was designed, with the following principles in mind:
-
- (a) The preference of 2′F-ANA and 2′F-ANA-2′F-RNA chimeras for the sense strand, and of 2′F-RNA for the antisense strand;
- (b) The low binding affinity of 1-1 altimers of 2′F-ANA and 2′F-RNA (duplexes jg-8 and jg-9 had Tm values 13-16° C. lower than the control sequence, see Table I;
- (c) The activity of a fully-modified 2′F-ANA sense strand was compared with that of a “fr-type” 2′F-ANA sense strand, which includes five RNA inserts near its 3′-end, when paired with a 2′F-RNA antisense strand.
- The resulting duplexes are presented in Table II. Each of two antisense strands (either RNA or 2′F-RNA) was paired with each of six modified sense strands (2′F-ANA or a 2′F-ANA-2′F-RNA chimera). The potency of these strands to induce RNAi was evaluated and the results are presented in
FIG. 3 . -
TABLE II Sequences of siRNAs targeting positions 515-533 of firefly luciferase with combinations of 2′F-ANA and 2′F-RNA. Name Description Sequence SEQ ID NO: kI- ctl RNA 5′-CGUACGCGGAAUACUUCGAtt-3′ 29 RNA 5′-UCGAAGUAUUCCGCGUACGtt-3′ 30 kI-1 2′F- RNA 5′- -3′ 31 RNA 5′-UCGAAGUAUUCCGCGUACGtt-3′ 30 kI-2 2′F- ANA 5′- CGTACGCGGAATACTTCGATT -3′ 32 RNA 5′-UCGAAGUAUUCCGCGUACGtt-3′ 30 kI-3 “fr” type 5′- CGTACGCGGAATAC UUCGA TT -3′ 33 RNA 5′-UCGAAGUAUUCCGCGUACGtt-3′ 30 kI-4 3-3 altimer 5′- CGT CGG ACT ATT -3′ 34 RNA 5′-UCGAAGUAUUCCGCGUACGtt-3′ 30 kI-5 3-3/1-1 alt 5′- CGT CGG A T C ATT -3′ 35 RNA 5′-UCGAAGUAUUCCGCGUACGtt-3′ 30 kI-6 1-1 altimer 5′-C T C C G A A T C ATT-3′ 36 RNA 5′-UCGAAGUAUUCCGCGUACGtt-3′ 30 kI-7 2′F- RNA 5′- -3′ 31 2′F- RNA 5′- -3′ 37 kI-8 2′F- ANA 5′- CGTACGCGGAATACTTCGATT -3′ 32 2′F- RNA 5′- -3′ 37 kI-9 V type 5′- CGTACGCGGAATAC UUCGA TT -3′ 33 2′F- RNA 5′- -3′ 37 kI-10 3-3 altimer 5′- CGT CGG ACT ATT -3′ 34 2′F- RNA 5′-p -3′ 37 kI-11 3-3/1-1 alt 5′- CGT CGG A T C ATT -3′ 35 2′F- RNA 5′-p -3′ 37 kI-12 1-1 altimer 5′- C T C C G A A T C ATT -3′ 36 2′F- RNA 5′- -3′ 37 Uppercase = RNA Lowercase = dna Uppercase bold underline = 2′F-ANA(FANA) Uppercase bold italic = 2′ p = 5′-Phosphate - Several results are clear from this set of duplexes. As shown in
FIG. 3 , nearly all of the duplexes are more effective the control siRNA. Four fully-modified duplexes (kl-7, kl-9, kl-10, kl-11) and five other heavily-modified duplexes (kl-4, kl-5, kl-6, kl-8, kl-12) have greater potency than the control for this second sequence of the firefly luciferase. - Furthermore, synergy between 2′F-RNA and 2′F-ANA is again visible. These duplexes can be thought of as belonging to two sub-series, the first with an RNA antisense strand (kl-1 to kl-6) and the second with a 2′F-RNA antisense strand (kl-7 to kl-12). Comparing the corresponding members of each series (kl-1 to kl-7, kl-2 to kl-8, etc), it is clear that all of the modified sense strands show better potency when paired to a 2′F-RNA antisense strand than an RNA antisense strand.
- Taking each sub-series separately, and ranking the duplexes in order of potency, a pattern can be observed: the sense strands follow the same order, with either antisense strand. Thus, the “worst” sense strand is all 2′F-ANA (kl-2 and kl-8), followed by the “fr-type” sense strand containing five RNA inserts (kl-3 and kl-9). It should be noted, however, that both kl-8 and kl-9 are nonetheless more potent than the control.
- Use of the chimeric 2′F-ANA-2′F-RNA sense strands led to better potency, again irrespective of the antisense strand used. The best sense strand was the 3-3/1-1 altimer strand (kl-5 and kl-11), suggesting that rational design for controlling thermodynamic bias does indeed improve potency. Duplex kl-11 was unsurpassed in both potency and efficacy. It is not possible even to estimate an IC50 value for this duplex, since at 2 nM, the lowest concentration used for these transfections, the silencing is still at its maximal level.
- Finally, it is worth noting that both duplexes kl-7 and kl-11 seem to be silencing at their maximum efficacy, since the dose response is essentially flat. The chimeric sense strand of kl-11 thus allows higher efficacy silencing (relative luciferase level of 0.12-0.15 instead of 0.21-0.24).
- As described herein, for example 2′F-ANA and 2′F-RNA can be combined in various ways in siRNA duplexes. For example, two types of combinations of these two modifications lead to increased potency: combining both chemistries in the sense strand, and combining an 2′F-RNA antisense strand with a 2′F-ANA or chimeric sense strand. Examples of both of these types of synergistic combinations led to increased potency.
- The sequences of the siRNAs used in the 4E-BP inhibition studies described herein are provided in Table III.
-
TABLE III Sequences of the siRNAs used in the 4E-BP inhibition studies described herein SEQ ID Sequence Oligo ID siRNA duplex ID NO: 5′ AACUCACCUGUGACCAAAAca 4EBP-1 HS Unmodified Control 1 5′ UUUUGGUCACAGGUGAGUUcc 4EBP-1 HAS 4EBP- 1 Human 2 5′ AAGACUCCAAAGUAGAAGUaa 4EBP-2 HS Unmodified Control 3 5′ ACUUCUACUUUGGAGUCUUca 4EBP-2 HAS 4EBP-2 Human and 4 Murine 5′ AACUCACCUGUGGCCAAAAca 4EBP-1 MS Unmodified Control 5 5′ UUUUGGCCACAGGUGAGUUcc 4EBP-1 MAS 4EBP-1 Murine 6 5′ AACTCACCTGTGGCCAAAACA 4EBP-1 MS_JG14 4EBP-1 Murine 147 5′ 4EBP-1 MAS_JG14 8 5′ AACTCACCTGTGACCAAAACA 4EBP-1 HS_JG14 4EBP-1 Human_14 9 5′ 4EBP-1 HAS_JG14 10 5′ AAGACTCCAAAGTAGAAGTAA 4EBP-2 MS_JG14 4EBP-2 Mouse_14 or 11 5′ 4EBP-2 MAS_JG14 4EBP-2 Human_14 12 5′ AAC CCT G C A ACA 4EBP-1 MS_611 4EBP1 Mouse_611 13 5′ 4EBP-1 MAS_611 14 5′ AAC CCT A C A ACA 4EBP-1 HS_611 4EBP1 Human_611 15 5′ 4EBP-1 HAS_611 16 5′ AAG CCA T G A TAA 4EBP-2 MS_611 4EBP2 Mouse_611 or 17 5′ 4EBP-2 MAS_611 4EBP2 Human_611 18 Uppercase = RNA Lowercase = dna Uppercase bold underline = 2′F-ANA (FANA) Uppercase bold italic = 2′ p = 5′-Phosphate - The results presented at
FIG. 4A indicate that unmodified siRNAs targeting human 4E-BP1 and 4E-BP2 are eliciting potent gene silencing (far right lanes in the two gels). As well, none of the scrambled (non-targeting) siRNAs affect expression levels of 4E-BP1 or 4E-BP2. Because Scrambled modifiedcontrol - The ability of chemically modified siRNAs to reproduce the 4E-BP1/2 double knockout phenotype was next determined using the HEK-Blue™ system according to the manufacturer's protocol (InvivoGen). The results of experiments performed to monitor the relative levels of
interferon 3 days post-siRNAs transfection and in the presence or absence of poly(I:C) are presented inFIG. 5 . When cells are treated with modified scrambled siRNA and poly(I:C), the relative IFN levels are similar to that of cells treated with unmodified scrambled sequence, showing the modification does not trigger a significant immunostimulatory response. In the case of treatment of cells with unmodified siRNAs targeting both 4E-BP1 and 2 at the same time, the relative levels of IFN in the cells increase to around 5 units in the absence of poly(I:C). When the cells were treated with poly(I:C) (a trigger of IFN production via RIG-I and MDA5 receptors), relative IFN levels are around 18, versus about 11 in scrambled siRNA treated cells, demonstrating that silencing 4E-BP1 and 2 increases the IFN response, similar to our observations in 4E-BP1/2 knockout mice. Finally, treatment with fully modified siRNA (corresponding to the —611 architecture) against 4E-BP1 and 2 in the presence of poly(I:C) results in relative IFN levels of about 42 units, which is a 4-fold increase as compared to scrambled treated cells, and a 2-fold increase as compared to cells treated with regular unmodified 4E-BP1 and 2 siRNA. - It was next tested whether 2′F-ANA could act synergistically with another RNA analog adopting northern sugar pucker, namely locked nucleic acid (LNA). LNA is locked into a rigid northern sugar conformation by a methylene bridge.
- The first series, referred to as “L-FL”, were designed by combining 2′F-ANA sense strands with antisense strands containing 2′F-ANA overhangs and LNA inserts at positions previously observed to have RNAi activity. The sequences of the duplexes of the L-FL series are provided in Table IV.
-
TABLE IV Sequences of the siRNAs of the L-FL series used in the experiments described herein Strand Sequence label siRNA label SEQ ID NO: 5′- GCTTGAAGTCTTTAATTAATT -3′ 303g L-FL1 27 5′- pUUAAUUAAAGACUUCAAGc GG -3′ GD2 38 5′- GCTTGAAGTCTTTAATTAATT -3′ 303g L-FL2 27 5′- pUUAAUUAAAGACUUCAaGc GG -3′ GD3 39 5′- GCTTGAAGTCTTTAATTAATT -3′ 303g L-FL3 27 5′- pUUAAUUaaAAGACUUCAAGc GG -3′ GD4 40 5′- GCTTGAAGTCTTTAATTAATT -3′ 303g L-FL4 27 5′- pUUAAUUAAAGACUUCAAGCgg-3′ 56 5′- GCTTGAAGTCTTTAATTAATT -3′ 303g L-FL5 27 5′- pUUAAUUAAAGACUUCAAGC GG -3′ GD1 42 5′- GCTTGAAGTCTTTA AUUAATT -3′ L-S-RF L-FL6 41 5′- pUUAAUUAAAGACUUCAAGc GG -3′ GD2 38 5′- GCTTGAAGTCTTTA AUUAA TT -3′ L-S-RF L-FL7 41 5′- pUUAAUUAAAGACUUCAaGc GG -3′ GD3 39 5′- GCTTGAAGTCTTTA AUUAA TT -3′ L-S-RF L-FL8 41 5′- pUUAAUUaaAAGACUUCAAGG GG -3′ GD4 40 5′- GCTTGAAGTCTTTA AUUAA TT -3′ L-S-RF L-FL9 41 5′- pUUAAUUAAAGACUUCAAGCgg-3′ 56 5′- GCTTGAAGTCTTTA AUUAA TT -3′ L-S-RF L-FL10 41 5′- pUUAAUUAAAGACUUCAAGC GG -3′ GD1 42 5′- GCUUGAAGUCUUUAAUUAAtt -3′ G1A L-FL11 19 5′- pUUAAUUAAAGACUUCAAGCgg-3′ 56 5′- GCUUGAAGUCUUUAAUUAAtt -3′ G1A L-FL12 19 5′- pUUAAUUAAAGACUUCAAGC GG -3′ GD1 42 5′- GCUUGAAGUCUUUAAUUAAtt -3′ G1A L-FL13 19 5′- pUUAAUUaaAAGACUUCAAGc GG -3′ GD4 40 5′- GCUUGAAGUCUUUAAUUAAtt -3′ G1A L-FL18 19 5′- UUAAUUAAAGACUUCAAGCgg -3′ G1B 20 5′- GCUUGAUUUCUGAAAUUAAtt -3′ 178H Sc Control 54 5′- UUAAUUUCAGAAAUCAAGCgg -3′ 178I 55 Uppercase = RNA Lowercase = dna Lowercase underline = Ina Uppercase bold underline = 2′F-ANA(FANA) Uppercase bold italic = 2′ p = 5′-Phosphate - The second series, referred to as “L-FL2”, was designed based on 2′F-ANA/2′F-RNA architectures shown to have significant potency-improving synergy (see Examples 2 and 3 above). The sequences of the duplexes of the L-FL2 series are provided at Table V.
-
TABLE V Sequences of the siRNAs of the L-FL2 series used in the studies described herein Strand Strands Labels siRNA labels SEQ ID NO: 5′- G CuU GAAG UCuUU AA uU AATT -3′ GD-21 L-FL2-1 43 5′- UUAAUUAAAGACUUCAAGCgg -3′ G1B 20 5′- G CuU GAAG UCuUU A A T u A A TT -3′ GD-22 L-FL2-2 44 5′- UUAAUUAAAGACUUCAAGCgg -3′ G1B 20 5′- G CuU GAAG UCuUU AA UU A A TT -3′ GD-23 L-FL2-3 45 5′- UUAAUUAAAGACUUCAAGCgg -3′ G1B 20 5′- G CuU GAAG UCuUU A A T U A A TT -3′ GD-24 L-FL2-4 46 5′- UUAAUUAAAGACUUCAAGCgg -3′ G1B 20 5′- G CuU GAAG UCuUU A AUUAA TT -3′ GD-25 L-FL2-5 47 5′- pUUAAUUAAAGACUUCAAGCgg -3′ G1B 20 5′- G CuU GAAG UCuUU AA uU AATT -3′ GD-21 L-FL2-6 43 5′- pUUAAUUAAAGACUUCAAGc GG -3′ GD2 38 5′- G CuU GAAG UCuUU A A T u A A TT -3′ GD-22 L-FL2-7 44 5′- pUUAAUUAAAGACUUCAAGc GG -3′ GD2 38 5′- G CuU GAAG UCuUU A AUUAA TT -3′ GD-23 L-FL2-8 45 5′- pUUAAUUAAAGACUUCAAGc GG -3′ GD2 38 5′- G CuU GAAG UCuUU A A T U A A TT -3′ GD-24 L-FL2-9 46 5′- pUUAAUUAAAGACUUCAAGc GG -3′ GD2 38 5′- G CuU GAAG UCuUU A AUUAA TT -3′ GD-25 L-FL2-10 47 5′- pUUAAUUAAAGACUUCAAGc GG -3′ GD2 38 5′- GCUUGAAGUCUUUAAUUAAtt -3′ G1A Control 19 5′- UUAAUUAAAGACUUCAAGCgg -3′ G1B 20 5′-GCUUGAUUUCUGAAAUUAAtt -3′ 178H Sc Control 54 5′- UUAAUUUCAGAAAUCAAGCgg -3′ 1781 55 Uppercase = RNA Lowercase = dna Lowercase underline = Ina Uppercase bold underline = 2′F-ANA(FANA) Uppercase bold italic = 2′ p = 5′-Phosphate - The third series, referred to as “L-FL3”, utilizes the same sense strands from L-FL2 annealed with all-2′F-RNA antisense strands. The sequences of the duplexes of the L-FL3 series are provided at Table VI.
-
TABLE VI Sequences of the siRNAs of the L-FL3 series used in the studies described herein Sequence Strand ID siRNA ID SEQ ID NO: 5′- G CuU GAAG UCuUU AA uUA ATT -3′ GD-21 L-FL3-1 43 5′- -3′ 303f 26 5′- G CuU GAAG UCuUU A A T u A A TT -3′ GD-22 L-FL3-2 44 5′- -3′ 303f 26 5′- G CuU GAAG UCuUU AA UU AATT -3′ GD-23 L-FL3-3 45 5′- -3′ 303f 26 5′- G CuU GAAG UCuUU A A T UAA TT -3′ GD-24 L-FL3-4 46 5′- -3′ 303f 26 5′- G CuU GAAG UCuUU A AUUAA TT -3′ GD-25 L-FL3-5 47 5′- -3′ 303f 26 Uppercase = RNA Lowercase = dna Lowercase underline = Ina Uppercase bold underline = 2′F-ANA(FANA) Uppercase bold italic = 2′ p = 5′-Phosphate - Each oligonucleotide was characterized by ESI-TOF mass spectroscopy (Table VII) and for some of the oligonucleotides by analytical denaturing PAGE followed by stains-all treatment.
-
TABLE VII Mass spectroscopy data for the oligonucleotides of the L-FL, L-FL2 and L-FL3 series Sequence Expected Mass (M − H)− Experimental Mass GD2 6814 6814.3 GD3 6826 6826.7 GD4 6814 6812.5 L-S-RF n.d. n.d. G1A 6618 6616.5 G1B 6674 6672.2 178H 6618 6616.4 178I 6674 6671.9 GD21 6707 6705.3 GD22 6720 6718 GD23 6696 6693 GD24 6708 6705.8 GD25 6690 6687.4 303g 6804 6802 303f 6911 6911 - Analysis of the data presented in
FIG. 6 indicates that antisense strand GD2, containing two 3′ FANA (2′F-ANA) overhangs followed by a single LNA residue is compatible with the RNAi machinery, and in some cases can improve siRNA potency relative to a regular RNA antisense strand (compare L-FL1 with L-FL4). Considering 3′-modified RNAs are generally more stable to nuclease degradation, this antisense architecture was chosen to move forward with in further studies, now focused on probing forintrastrand 2′F-ANA/LNA synergy in the sense strand. - As shown above (Examples 2 and 3), potent gene silencing may be achieved using 2′F-ANA/2′F-RNA chimera siRNAs. Chimeric 2′F-ANA/LNA siRNA architectures comprising the L-FL2 series of siRNAs were then designed and studied. Sense strands were designed with alternating regions of 2′F-ANA and LNA moving from 5′ to 3′. LNA incorporation was kept to a minimum by surrounding strongly northern-puckered LNA inserts with RNA. Chemical modifications at the 3′ ends of the sense strands were varied in attempts to capitalize on the observed thermodynamic bias of RISC for loading of the siRNA strand with the weakest binding affinity at the 5′ end.7 Sense strands GD21-GD25 are identical until
nucleotide 14, after which several patterns of chemical modification were employed. Strands GD21 and 22 feature alternating LNA-2′F-ANA regions designed to explore the effects of placing contrasting sugar puckers (northern vs. southeastern) side by side in a sense strand. GD23-25 feature various patterns of 2′F-ANA modification combined with unmodified RNA, including 1-1 altimer designs, 2-2 altimer designs, and fullyRNA 3′ regions followed by 2′F-ANA overhangs. The Tm of the oligonucleotide duplexes of the L-FL2 series is provided in Table VIII below. -
TABLE VIII Tm of the oligonucleotide duplexes of the L-FL2 series siRNA Tm (° C.) L-FL2-1 62.9 L-FL2-2 59.2 L-FL2-3 58.4 L-FL2-4 55.0 L-FL2-5 58.6 L-FL2-6 65.7 L-FL2-7 62.2 L-FL2-8 n.d. L-FL2-9 60.5 L-FL2-10 61.9 Control 60.5 - According to the Tm data obtained, the 3′ chemical modifications did not create significant changes in duplex binding affinity, suggesting that strand bias for loading of the proper antisense strand was not introduced. However, the siRNA sequence has high A:U content at the 5′ end of the antisense strand, favoring proper RISC loading, and perhaps further strand bias is unnecessary. To examine the gene silencing activity of these LNA/2′F-ANA sense strands, siRNAs were prepared by annealing GD21-GD25 with either a regular RNA antisense strand, or with GD2, the potent LNA/2′F-ANA antisense strand from the L-FL series. Indeed, despite the failure to introduce significant strand bias, several of these modified architectures were able to elicit potent gene silencing, comparable to or better than the native RISC substrate, dsRNA.
- Initial siRNA assays with the L-FL2 series indicated potency increases several fold better than unmodified controls. In fact, 70-90% knockdown was observed at subnanomolar ranges for L-FL2-9 and L-FL2-10, stronger knockdown than even 2 nM treatments with unmodified siRNA. Subsequent firefly luciferase knockdown assays indicate potent knockdown from the L-FL2 series. Shown in
FIG. 7 are the knockdown results for the best siRNAs in the L-FL2 series. Several of the architectures are well tolerated by the RISC machinery. Some of the architectures tested here are much more potent gene silencers than unmodified siRNA, especially at lower doses. Additionally, the LNA-containing designs appear to be more potent than one of the potent 2′F-ANA/2′F-RNA siRNA designs described above (jg-14). These data thus show that heavily modified siRNA designs have seemingly no detrimental effects on gene silencing. - The L-FL2 series demonstrates sense stand modification plans that are highly compatible with gene knockdown. However, in these cases the antisense strand remains unmodified, or only 3′-modified. It was next tested whether it was possible to combine these potent sense strand architectures with antisense strand modifications compatible with RISC, such as 2′F-RNA antisense strands.
- Based on the efficacy of the LNA/2′F-ANA sense strands from the L-FL2 series, and on the observed efficacy of fully 2′F-RNA antisense strands, highly modified siRNAs containing only 7-11 RNA inserts were designed (L-FL3 series). These chemical modification architectures represent the combination of the designs shown herein to be compatible with siRNA-based silencing (Examples 2 and 3, L-FL1 and L-FL2 series). As shown in
FIG. 8 , these heavily modified siRNA-mimics show potent gene-silencing abilities. Some of these modified siRNAs are significantly more potent than control siRNA, even at the midrange 0.08 nM dose where the potency of the modified siRNAs of the L-FL2 series was about equal to that of the unmodified siRNA. - C-myb is a protooncogene implicated in leukemia. It encodes proteins essential for hematopoetic cell proliferation. 2′F-ANA/2′F-RNA and 2′F-ANA/2′F-RNA/LNA architectures shown to have luciferase and/or 4E-BP gene silencing activities were tested against another target, namely c-myb. The sequences of the duplexes of the C-myb series are provided in Table IX.
-
TABLE IX Sequences of the siRNAs of the C-myb series used in the studies described herein Strands siRNA labels SEQ ID NO: 5′- UGUUAUUGCCAAGCACUUAAA -3′ Cmyb-1 48 5′- UAAGUGCUUGGCAAUAACAGA -3′ 49 5′- TGT TGC G A T AAA -3′ Cmyb-2 50 5′- p -3′ 51 5′- T GuU ATTG CCaAG CA cU TAAA -3′ Cmyb-3 52 5′- p -3′ 51 5′- TGT TGC G A T AAA -3′ Cymb-4 50 5′- UAAGUGCUUGGCAAUAACAGA -3′ 49 5′- T GuU ATTG CCaAG CA cU TAAA -3′ Cmyb-5 52 5′- UAAGUGCUUGGCAAUAACAGA -3′ 49 5′- UGUUAUUGCCAAGCACUUAAA -3′ Cmyb-6 48 5′- p -3′ 51 5′- GCUUGAAGUCUUUAAUUAAtt -3′ Scrambled 19 5′- UUAAUUAAAGACUUCAAGCgg -3′ 20 5′- CGT CGG A T C ATT -3′ Scrambled 35 5′- -3′ Mod. 1 37 5′- G CuU GAAG UCuUU AA uU AATT -3′ Scrambled 43 5′- -3′ Mod. 2 26 Uppercase = RNA Lowercase = dna Lowercase underline = Ina Uppercase bold underline = 2′F-ANA(FANA) Uppercase bold italic = 2′ p = 5′-Phosphate - As shown in
FIG. 9A , 2′F-ANA/2′F-RNA and 2′F-ANA/2′F-RNA/LNA modified siRNA are capable of silencing gene expression in another target, and are better at silencing c-myb than unmodified siRNA at the lower dosages. The 2′F-ANA/2′F-RNA architecture appears to be more potent under the experimental conditions tested. -
FIG. 9B shows the survival rate (y-axis represents number of leukemia cells still living after the indicated time periods after treatment with siRNA designed to target c-myb and prevent leukemia cell proliferation) following siRNA treatment. Interestingly, unmodified siRNA-treated leukemia cells rebound 6 days after treatment and start proliferating again, whereas several of the modified siRNAs still prevent proliferation after 6 days. This suggests that modified siRNAs are not degraded as much as unmodified siRNAs after these time periods. - The novel chimeric siRNA architectures reported herein represent previously unexplored siRNA-mimics capable of equivalent or improved potencies compared to unmodified siRNA.
-
TABLE X Summary of siRNAs used in the studies described herein Sequence siRNA duplex ID SEQ ID NO: siRNAs of Table II 5′ AACUCACCUGUGACCAAAAca Unmodified Control 1 5′ UUUUGGUCACAGGUGAGUUcc 4EBP- 1 Human 2 5′ AAGACUCCAAAGUAGAAGUaa Unmodified Control 3 5′ ACUUCUACUUUGGAGUCUUca 4EBP-2 Human 4 and Murine 5′ AACUCACCUGUGGCCAAAAca Unmodified Control 5 5′ UUUUGGCCACAGGUGAGUUcc 4EBP-1 Murine 6 5′ AACTCACCTGTGGCCAAAACA 4EBP-1 Murine_14 7 5′ 8 5′ AACTCACCTGTGACCAAAACA 4EBP-1 Human_14 9 5′ 10 5′ AAGACTCCAAAGTAGAAGTAA 4EBP-2 Mouse_14 or 11 5′ 4EBP-2 Human_14 12 5′ AAC CCT G C A ACA 4EBP1 Mouse_611 13 5′ 14 5′ AAC CCT A C A ACA 4EBP1 15 5′ Human_611 16 5′ AAG CCA T G A TAA 4EBP2 Mouse_611 or 17 5′ 4EBP2 18 Human_611 siRNAs of Table I 5′- GCUUGAAGUCUUUAAUUAAU-3′ jg-1 19 5′- UUAAUUAAAGACUUCAAGCgg-3′ 20 5′- G GAAG AA AATT -3′ jg-2 21 5′- A AA AAGA AAG GG -3′ 22 5′- G T G A T T T A T ATT -3′ jg-3 23 5′- T A T A G C T A G GG -3′ 24 5′- -3′ jg-4 25 5′-p -3′ 26 5′- GCTTGAAGTCTTTAATTAATT -3′ jg-5 27 5′-p TTAATTAAAGACT T CAAGCGG -3′ 28 5′- G GAAG AA AATT -3′ jg-6 21 5′- UUAAUUAAAGACUUCAAGCgg-3′ 20 5′- GCUUGAAGUCUUUAAUUAAtt-3′ jg-7 19 5′- AA AAAGA AAG GG -3′ 22 5′- G T G A T T T A T ATT -3′ j9-8 23 5′- UUAAUUAAAGACUUCAAGCgg-3′ 20 5′- GCUUGAAGUCUUUAAUUAAtt-3′ jg-9 19 5′- T A T A G C T A G GG -3 24 5′- -3′ jg-10 25 5′- UUAAUUAAAGACUUCAAGCgg-3′ 20 5′- GCUUGAAGUCUUUAAUUAAtt-3′ jg-11 19 5′- -3′ 26 5′- GCTTGAAGTCTTTAATTAATT -3′ jg-12 27 5′- UUAAUUAAAGACUUCAAGCgg-3′ 20 5′- GCUUGAAGUCUUUAAUUAAtt-3′ jg-13 19 5′-p TTAATTAAAGACTTCAAGCGG -3′ 28 5′- GCTTGAAGTCTTTAATTAATT -3′ jg-14 27 5′- -3′ 26 5′- -3′ jg-15 25 5′-p TTAATTAAAGACTTCAAGCGG -3′ 28 siRNAs of Table 11 5′- CGUACGCGGAAUACUUCGAtt-3′ kI-ctl 29 5′- UCGAAGUAUUCCGCGUACGtt-3′ 30 5′- -3′ kI-1 31 5′- UCGAAGUAUUCCGCGUACGtt-3′ 30 5′- CGTACGCGGAATACTTCGATT -3′ kI-2 32 5′- UCGAAGUAUUCCGCGUACGtt-3′ 30 5'- CGTACGCGGAATAC UUCGA TT -3′ kI-3 33 5′- UCGAAGUAUUCCGCGUACGtt-3′ 30 5′- CGT CGG ACT ATT-3′ kI-4 34 5′- UCGAAGUAUUCCGCGUACGtt-3′ 30 5′- CGT CGG A T C ATT -3′ kI-5 35 5′- UCGAAGUAUUCCGCGUACGtt-3′ 30 5′- C T C C G A A T C ATT -3′ kI-6 36 5′- UCGAAGUAUUCCGCGUACGtt-3′ 30 5′- -3′ kI-7 31 5′- -3′ 37 5′- CGTACGCGGAATACT T CGATT -3′ kI-8 32 5′- -3′ 37 5′- CGTACGCGGAATAC UUCGA TT -3′ kI-9 33 5′- -3′ 37 5′- CGT CGG ACT ATT -3′ kI-10 34 5′- -3′ 37 5′- CGT CGG A T C ATT -3′ kI-11 35 5′- -3′ 37 5′- C T C C G A A T C ATT -3′ kI-12 36 5′- -3′ 37 siRNAs of Table IV 5′- GCTTGAAGTCTTTAATTAATT -3′ L-FL1 27 5′-pUUAAUUAAAGACUUCAAGc GG -3′ 38 5′- GCTTGAAGTCTTTAATTAATT -3′ L-FL2 27 5′-pUUAAUUAAAGACUUCAaGc GG -3′ 39 5′- GCTTGAAGTCTTTAATTAATT -3′ L-FL3 27 5′-pUUAAUUaaAAGACUUCAAGc GG -3′ 40 5′- GCTTGAAGTCTTTA AUUAA TT -3′ L-FL6 41 5′-pUUAAUUAAAGACUUCAAGc GG -3′ 38 5′- GCTTGAAGTCTTTA AUUAA TT -3′ L-FL7 41 5′-pUUAAUUAAAGACUUCAaGc GG -3′ 39 5′- GCTTGAAGTCTTTA AUUAA TT -3′ L-FL8 41 5′-pUUAAUUaaAAGACUUCAAGc GG -3′ 40 5′- GCUUGAAGUCUUUAAUUAAtt-3′ L-FL13 19 5′-pUUAAUUAAAGACUUCAAGc GG -3′ 38 5′- GCUUGAAGUCUUUAAUUAAtt-3′ L-FL14 19 5′-pUUAAUUAAAGACUUCAaGc GG -3′ 39 5′- GCUUGAAGUCUUUAAUUAAtt-3′ L-FL15 19 5′-pUUAAUUaaAAGACUUCAAGc GG -3′ 40 5′- GCUUGAAGUCUUUAAUUAAtt-3′ L-FL12 19 5′-pUUAAUUAAAGACUUCAAGC GG -3′ 42 5′- GCUUGAAGUCUUUAAUUAAtt-3′ Control 19 5′-UUAAUUAAAGACUUCAAGCgg-3′ 20 5′-GCUUGAUUUCUGAAAUUAAtt-3′ Sc control 54 5′-UUAAUUUCAGAAAUCAAGCgg-3′ 55 siRNAs of Table V 5′- GCTTGAAGTCTTTAATTAATT -3′ L-FL1 27 5′-pUUAAUUAAAGACUUCAAGc GG -3′ 38 5′- GCTTGAAGTCTTTAATTAATT -3′ L-FL2 27 5′-pUUAAUUAAAGACUUCAaGc GG -3′ 39 5′- GCTTGAAGTCTTTAATTAATT -3′ L-FL3 27 5′-pUUAAUUaaAAGACUUCAAGc GG -3′ 40 5′- GCTTGAAGTCTTTAATTAATT -3′ L-FL4 27 5′-pUUAAUUAAAGACUUCAAGCgg-3′ 56 5′- GCTTGAAGTCTTTAATTAATT -3′ L-FL5 27 5′-pUUAAUUAAAGACUUCAAGC GG -3′ 42 5′- GCTTGAAGTCTTTA AUUAA TT -3′ L-FL6 41 5′-pUUAAUUAAAGACUUCAAGc GG -3′ 38 5′- GCTTGAAGTCTTTA AUUAA TT -3′ L-FL7 41 5′-pUUAAUUAAAGACUUCAaGc GG -3′ 39 5′- GCTTGAAGTCTTTA AUUAA TT -3′ L-FL8 41 5′-pUUAAUUaaAAGACUUCAAGc GG -3′ 40 5′- GCTTGAAGTCTTTA AUUAA TT -3′ L-FL9 41 5′-pUUAAUUAAAGACUUCAAGCgg-3′ 56 5′- GCTTGAAGTCTTTA AUUAA TT -3′ L-FL10 41 5′-pUUAAUUAAAGACUUCAAGC GG -3′ 42 5′- GCUUGAAGUCUUUAAUUAAtt-3′ L-FL11 19 5′-pUUAAUUAAAGACUUCAAGCgg-3′ 56 5′- GCUUGAAGUCUUUAAUUAAtt-3′ L-FL12 19 5′-pUUAAUUAAAGACUUCAAGC GG -3′ 42 5′- GCUUGAAGUCUUUAAUUAAtt-3′ L-FL13 19 5′-pUUAAUUaaAAGACUUCAAGc GG -3′ 40 5′- GCUUGAAGUCUUUAAUUAAtt-3′ L-FL18 19 5′-UUAAUUAAAGACUUCAAGCgg-3′ 20 5′-GCUUGAUUUCUGAAAUUAAtt-3′ Sc Control 54 5′-UUAAUUUCAGAAAUCAAGCgg-3′ 55 siRNAS of Table VI 5′- G CuU GAAG UCuUU AA uU AATT -3′ L-FL3-1 43 5′- -3′ 26 5′- G CuU GAAG UCuUU A A T u A A TT -3′ L-FL3-2 44 5′- -3′ 26 5′- G CuU GAAG UCuUU AA UU AATT -3′ L-FL3-3 45 5′- -3′ 26 5′- G CuU GAAG UCuUU A A T U A A TT -3′ L-FL3-4 46 5′- -3′ 26 5′- G CuU GAAG UCuUU A AUUAA TT -3′ L-FL3-5 47 5′- -3′ 26 siRNAs of Table IX 5′- UGUUAUUGCCAAGCACUUAAA-3′ Cmyb-1 48 5′-UAAGUGCUUGGCAAUAACAGA-3′ 49 5′- TGT TGC G A T AAA -3′ Cmyb-2 50 5′- -3′ 51 5′- T GuU ATTG CCaAG CA cU TAAA -3′ Cmyb-3 52 5′- -3′ 51 5′- TGT TGC G A T AAA-3′ Cmyb-4 50 5′-UAAGUGCUUGGCAAUAACAGA-3′ 49 5′- T GuU ATTG CCaAG CA cU TAAA -3′ Cm yb-5 52 5′-UAAGUGCUUGGCAAUAACAGA-3′ 49 5′- UGUUAUUGCCAAGCACUUAAA-3′ Cmyb-6 48 5′- -3′ 51 5′- GCUUGAAGUCUUUAAUUAAtt-3′ Sc 19 5′-UUAAUUAAAGACUUCAAGCgg-3′ 20 5′- CGT CGG A T C ATT-3 ′ Sc Mod 1 35 5′- -3′ 37 5′- G CuU GAAG UCuUU AA uU AATT -3 ′ Sc Mod 2 43 5′- -3′ 26 siRNAs of FIG. 4 5′ AACUCACCUGUGACCAAAAca Unmodified Control 1 5′ UUUUGGUCACAGGUGAGUUcc 4EBP-1 Human 2 (siRNA Control 1) 5′ AACTCACCTGTGACCAAAACA 4EBP-1 Human_14 9 5′ 10 5′ AAC CCT A C A ACA 4EBP1 15 5′ Human_611 16 5′ AAGACUCCAAAGUAGAAGUaa Unmodified Control 3 5′ ACUUCUACUUUGGAGUCUUca 4EBP-2 Human 4 (siRNA Control 2) 5′ AAGACTCCAAAGTAGAAGTAA 4EBP-2 Human_14 11 5′ 12 5′ AAG CCA T G A TAA 4EBP2 17 5′ Human_611 18 5′- GCUUGAAGUCUUUAAUUAAtt-3′ Scram bled (Sc) 19 5′-UUAAUUAAAGACUUCAAGCgg-3 ′ Control 20 5′- GCTTGAAGTCTTTAATTAATT -3′ Scrambled (Sc) 27 5′- -3′ Modified Control 153 5′- CGT CGG A T C ATT -3′ Scrambled (Sc) 35 5′- -3′ Modified Control 237 Uppercase = RNA Lowercase = dna Lowercase underline = Ina Uppercase bold underline = 2′F-ANA(FANA) Uppercase bold italic = 2′ p = 5′-Phosphate - Although the present invention has been described hereinabove by way of specific embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims. In the claims, the word “comprising” is used as an open-ended term, substantially equivalent to the phrase “including, but not limited to”. The singular forms “a”, “an” and “the” include corresponding plural references unless the context clearly dictates otherwise.
Claims (23)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US12/996,362 US9090649B2 (en) | 2008-06-05 | 2009-06-05 | Oligonucleotide duplexes comprising DNA-like and RNA-like nucleotides and uses thereof |
Applications Claiming Priority (8)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US5918608P | 2008-06-05 | 2008-06-05 | |
CA002635187A CA2635187A1 (en) | 2008-06-05 | 2008-06-17 | Oligonucleotide duplexes and uses thereof |
CA2635187 | 2008-06-17 | ||
CAPCTCA2008002259 | 2008-12-19 | ||
WOPCT/CA2008/002259 | 2008-12-19 | ||
PCT/CA2008/002259 WO2009076775A1 (en) | 2007-12-19 | 2008-12-19 | Immune response modulation and uses thereof |
US12/996,362 US9090649B2 (en) | 2008-06-05 | 2009-06-05 | Oligonucleotide duplexes comprising DNA-like and RNA-like nucleotides and uses thereof |
PCT/CA2009/000789 WO2009146556A1 (en) | 2008-06-05 | 2009-06-05 | Oligonucleotide duplexes comprising dna-like and rna-like nucleotides and uses thereof |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/CA2009/000789 A-371-Of-International WO2009146556A1 (en) | 2008-06-05 | 2009-06-05 | Oligonucleotide duplexes comprising dna-like and rna-like nucleotides and uses thereof |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US14/810,148 Continuation US9719091B2 (en) | 2008-06-05 | 2015-07-27 | Oligonucleotide duplexes comprising DNA-like and RNA-like nucleotides and uses thereof |
Publications (2)
Publication Number | Publication Date |
---|---|
US20110077286A1 true US20110077286A1 (en) | 2011-03-31 |
US9090649B2 US9090649B2 (en) | 2015-07-28 |
Family
ID=41397419
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US12/996,362 Active US9090649B2 (en) | 2008-06-05 | 2009-06-05 | Oligonucleotide duplexes comprising DNA-like and RNA-like nucleotides and uses thereof |
US14/810,148 Active US9719091B2 (en) | 2008-06-05 | 2015-07-27 | Oligonucleotide duplexes comprising DNA-like and RNA-like nucleotides and uses thereof |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US14/810,148 Active US9719091B2 (en) | 2008-06-05 | 2015-07-27 | Oligonucleotide duplexes comprising DNA-like and RNA-like nucleotides and uses thereof |
Country Status (6)
Country | Link |
---|---|
US (2) | US9090649B2 (en) |
EP (1) | EP2294195B1 (en) |
JP (1) | JP5684116B2 (en) |
CA (2) | CA2635187A1 (en) |
ES (1) | ES2643576T3 (en) |
WO (1) | WO2009146556A1 (en) |
Cited By (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9328346B2 (en) | 2010-11-12 | 2016-05-03 | The General Hospital Corporation | Polycomb-associated non-coding RNAs |
US9580708B2 (en) | 2011-09-14 | 2017-02-28 | Rana Therapeutics, Inc. | Multimeric oligonucleotides compounds |
US9790494B2 (en) | 2012-09-14 | 2017-10-17 | Translate Bio Ma, Inc. | Multimeric oligonucleotide compounds having non-nucleotide based cleavable linkers |
US9920317B2 (en) | 2010-11-12 | 2018-03-20 | The General Hospital Corporation | Polycomb-associated non-coding RNAs |
US10059941B2 (en) | 2012-05-16 | 2018-08-28 | Translate Bio Ma, Inc. | Compositions and methods for modulating SMN gene family expression |
US10058623B2 (en) | 2012-05-16 | 2018-08-28 | Translate Bio Ma, Inc. | Compositions and methods for modulating UTRN expression |
US10174315B2 (en) | 2012-05-16 | 2019-01-08 | The General Hospital Corporation | Compositions and methods for modulating hemoglobin gene family expression |
US10174328B2 (en) | 2013-10-04 | 2019-01-08 | Translate Bio Ma, Inc. | Compositions and methods for treating amyotrophic lateral sclerosis |
US10174323B2 (en) | 2012-05-16 | 2019-01-08 | The General Hospital Corporation | Compositions and methods for modulating ATP2A2 expression |
US10655128B2 (en) | 2012-05-16 | 2020-05-19 | Translate Bio Ma, Inc. | Compositions and methods for modulating MECP2 expression |
US10758558B2 (en) | 2015-02-13 | 2020-09-01 | Translate Bio Ma, Inc. | Hybrid oligonucleotides and uses thereof |
US10837014B2 (en) | 2012-05-16 | 2020-11-17 | Translate Bio Ma, Inc. | Compositions and methods for modulating SMN gene family expression |
US10858650B2 (en) | 2014-10-30 | 2020-12-08 | The General Hospital Corporation | Methods for modulating ATRX-dependent gene repression |
US10900036B2 (en) | 2015-03-17 | 2021-01-26 | The General Hospital Corporation | RNA interactome of polycomb repressive complex 1 (PRC1) |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP4082551A1 (en) | 2006-08-08 | 2022-11-02 | Rheinische Friedrich-Wilhelms-Universität Bonn | Structure and use of 5' phosphate oligonucleotides |
EP2297323A1 (en) | 2008-05-21 | 2011-03-23 | Hartmann, Gunther | 5' triphosphate oligonucleotide with blunt end and uses thereof |
CA2635187A1 (en) | 2008-06-05 | 2009-12-05 | The Royal Institution For The Advancement Of Learning/Mcgill University | Oligonucleotide duplexes and uses thereof |
EP2508530A1 (en) | 2011-03-28 | 2012-10-10 | Rheinische Friedrich-Wilhelms-Universität Bonn | Purification of triphosphorylated oligonucleotides using capture tags |
EP2712870A1 (en) | 2012-09-27 | 2014-04-02 | Rheinische Friedrich-Wilhelms-Universität Bonn | Novel RIG-I ligands and methods for producing them |
US20230159922A1 (en) * | 2019-08-15 | 2023-05-25 | Ionis Pharmaceuticals, Inc. | Modified oligomeric compounds and uses thereof |
Citations (22)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5539082A (en) * | 1993-04-26 | 1996-07-23 | Nielsen; Peter E. | Peptide nucleic acids |
US5580859A (en) * | 1989-03-21 | 1996-12-03 | Vical Incorporated | Delivery of exogenous DNA sequences in a mammal |
US5593972A (en) * | 1993-01-26 | 1997-01-14 | The Wistar Institute | Genetic immunization |
US6083482A (en) * | 1999-05-11 | 2000-07-04 | Icn Pharmaceuticals, Inc. | Conformationally locked nucleosides and oligonucleotides |
US6336859B2 (en) * | 1993-03-31 | 2002-01-08 | Progressive Games, Inc. | Method for progressive jackpot gaming |
US20020132788A1 (en) * | 2000-11-06 | 2002-09-19 | David Lewis | Inhibition of gene expression by delivery of small interfering RNA to post-embryonic animal cells in vivo |
US20020173478A1 (en) * | 2000-11-14 | 2002-11-21 | The Trustees Of The University Of Pennsylvania | Post-transcriptional gene silencing by RNAi in mammalian cells |
US20030143732A1 (en) * | 2001-04-05 | 2003-07-31 | Kathy Fosnaugh | RNA interference mediated inhibition of adenosine A1 receptor (ADORA1) gene expression using short interfering RNA |
US6639059B1 (en) * | 1999-03-24 | 2003-10-28 | Exiqon A/S | Synthesis of [2.2.1]bicyclo nucleosides |
US6734291B2 (en) * | 1999-03-24 | 2004-05-11 | Exiqon A/S | Synthesis of [2.2.1]bicyclo nucleosides |
US6780428B2 (en) * | 2001-06-08 | 2004-08-24 | Labopharm, Inc. | Unimolecular polymeric micelles with an ionizable inner core |
US6780324B2 (en) * | 2002-03-18 | 2004-08-24 | Labopharm, Inc. | Preparation of sterile stabilized nanodispersions |
US20040180351A1 (en) * | 2002-08-05 | 2004-09-16 | Atugen Ag | Interfering RNA molecules |
US6939564B2 (en) * | 2001-06-08 | 2005-09-06 | Labopharm, Inc. | Water-soluble stabilized self-assembled polyelectrolytes |
US20050196787A1 (en) * | 2004-01-22 | 2005-09-08 | Sanjay Bhanot | Modulation of eIF4E-BP2 expression |
US7018655B2 (en) * | 2002-03-18 | 2006-03-28 | Labopharm, Inc. | Amphiphilic diblock, triblock and star-block copolymers and their pharmaceutical compositions |
US7053207B2 (en) * | 1999-05-04 | 2006-05-30 | Exiqon A/S | L-ribo-LNA analogues |
US7084125B2 (en) * | 1999-03-18 | 2006-08-01 | Exiqon A/S | Xylo-LNA analogues |
US7094810B2 (en) * | 2001-06-08 | 2006-08-22 | Labopharm, Inc. | pH-sensitive block copolymers for pharmaceutical compositions |
US20060198891A1 (en) * | 2004-11-29 | 2006-09-07 | Francois Ravenelle | Solid formulations of liquid biologically active agents |
US7262253B2 (en) * | 2003-12-02 | 2007-08-28 | Labopharm, Inc. | Process for the preparation of amphiphilic poly (N-vinyl-2-pyrrolidone) block copolymers |
US20090258071A1 (en) * | 2006-09-22 | 2009-10-15 | Labopharm, Inc. | Compositions and methods for ph targeted drug delivery |
Family Cites Families (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5034506A (en) | 1985-03-15 | 1991-07-23 | Anti-Gene Development Group | Uncharged morpholino-based polymers having achiral intersubunit linkages |
US6338859B1 (en) | 2000-06-29 | 2002-01-15 | Labopharm Inc. | Polymeric micelle compositions |
AU2004221760B2 (en) | 2003-03-21 | 2010-03-18 | Roche Innovation Center Copenhagen A/S | Short interfering RNA (siRNA) analogues |
CA2622761A1 (en) * | 2005-09-16 | 2007-03-22 | Coley Pharmaceutical Gmbh | Modulation of immunostimulatory properties of short interfering ribonucleic acid (sirna) by nucleotide modification |
MX2008005508A (en) * | 2005-10-28 | 2008-11-18 | Topigen Pharmaceuticals Inc | Small interfering ribonucleic acid duplexes comprising arabinose modified nucleotides. |
US20090069263A1 (en) * | 2005-12-16 | 2009-03-12 | Damha Masad J | 4'-thioarabinonucleotide-containing oligonucleotides, compounds and methods for their preparation and uses thereof |
EP2052079A2 (en) | 2006-07-17 | 2009-04-29 | Sirna Therapeutics Inc. | Rna interference mediated inhibition of proprotein convertase subtilisin kexin 9 (pcsk9) gene expression using short interfering nucleic acid (sina) |
CA2635187A1 (en) | 2008-06-05 | 2009-12-05 | The Royal Institution For The Advancement Of Learning/Mcgill University | Oligonucleotide duplexes and uses thereof |
-
2008
- 2008-06-17 CA CA002635187A patent/CA2635187A1/en not_active Abandoned
-
2009
- 2009-06-05 EP EP09757018.8A patent/EP2294195B1/en active Active
- 2009-06-05 WO PCT/CA2009/000789 patent/WO2009146556A1/en active Application Filing
- 2009-06-05 US US12/996,362 patent/US9090649B2/en active Active
- 2009-06-05 ES ES09757018.8T patent/ES2643576T3/en active Active
- 2009-06-05 CA CA2764456A patent/CA2764456C/en active Active
- 2009-06-05 JP JP2011511949A patent/JP5684116B2/en active Active
-
2015
- 2015-07-27 US US14/810,148 patent/US9719091B2/en active Active
Patent Citations (25)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5580859A (en) * | 1989-03-21 | 1996-12-03 | Vical Incorporated | Delivery of exogenous DNA sequences in a mammal |
US5589466A (en) * | 1989-03-21 | 1996-12-31 | Vical Incorporated | Induction of a protective immune response in a mammal by injecting a DNA sequence |
US5593972A (en) * | 1993-01-26 | 1997-01-14 | The Wistar Institute | Genetic immunization |
US6336859B2 (en) * | 1993-03-31 | 2002-01-08 | Progressive Games, Inc. | Method for progressive jackpot gaming |
US5539082A (en) * | 1993-04-26 | 1996-07-23 | Nielsen; Peter E. | Peptide nucleic acids |
US7084125B2 (en) * | 1999-03-18 | 2006-08-01 | Exiqon A/S | Xylo-LNA analogues |
US6734291B2 (en) * | 1999-03-24 | 2004-05-11 | Exiqon A/S | Synthesis of [2.2.1]bicyclo nucleosides |
US6639059B1 (en) * | 1999-03-24 | 2003-10-28 | Exiqon A/S | Synthesis of [2.2.1]bicyclo nucleosides |
US7053207B2 (en) * | 1999-05-04 | 2006-05-30 | Exiqon A/S | L-ribo-LNA analogues |
US6083482A (en) * | 1999-05-11 | 2000-07-04 | Icn Pharmaceuticals, Inc. | Conformationally locked nucleosides and oligonucleotides |
US20020132788A1 (en) * | 2000-11-06 | 2002-09-19 | David Lewis | Inhibition of gene expression by delivery of small interfering RNA to post-embryonic animal cells in vivo |
US20020173478A1 (en) * | 2000-11-14 | 2002-11-21 | The Trustees Of The University Of Pennsylvania | Post-transcriptional gene silencing by RNAi in mammalian cells |
US20030143732A1 (en) * | 2001-04-05 | 2003-07-31 | Kathy Fosnaugh | RNA interference mediated inhibition of adenosine A1 receptor (ADORA1) gene expression using short interfering RNA |
US6939564B2 (en) * | 2001-06-08 | 2005-09-06 | Labopharm, Inc. | Water-soluble stabilized self-assembled polyelectrolytes |
US6780428B2 (en) * | 2001-06-08 | 2004-08-24 | Labopharm, Inc. | Unimolecular polymeric micelles with an ionizable inner core |
US7094810B2 (en) * | 2001-06-08 | 2006-08-22 | Labopharm, Inc. | pH-sensitive block copolymers for pharmaceutical compositions |
US7510731B2 (en) * | 2001-06-08 | 2009-03-31 | Labopharm Inc. | Water-soluble stabilized self-assembled polyelectrolytes |
US7018655B2 (en) * | 2002-03-18 | 2006-03-28 | Labopharm, Inc. | Amphiphilic diblock, triblock and star-block copolymers and their pharmaceutical compositions |
US6780324B2 (en) * | 2002-03-18 | 2004-08-24 | Labopharm, Inc. | Preparation of sterile stabilized nanodispersions |
US20040180351A1 (en) * | 2002-08-05 | 2004-09-16 | Atugen Ag | Interfering RNA molecules |
US7262253B2 (en) * | 2003-12-02 | 2007-08-28 | Labopharm, Inc. | Process for the preparation of amphiphilic poly (N-vinyl-2-pyrrolidone) block copolymers |
US7838600B2 (en) * | 2003-12-02 | 2010-11-23 | Labopharm, Inc. | Process for the preparation of amphiphilic poly(N-vinyl-2-pyrrolidone) block copolymers |
US20050196787A1 (en) * | 2004-01-22 | 2005-09-08 | Sanjay Bhanot | Modulation of eIF4E-BP2 expression |
US20060198891A1 (en) * | 2004-11-29 | 2006-09-07 | Francois Ravenelle | Solid formulations of liquid biologically active agents |
US20090258071A1 (en) * | 2006-09-22 | 2009-10-15 | Labopharm, Inc. | Compositions and methods for ph targeted drug delivery |
Cited By (26)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10119144B2 (en) | 2010-11-12 | 2018-11-06 | The General Hospital Corporation | Polycomb-associated non-coding RNAs |
US10053694B2 (en) | 2010-11-12 | 2018-08-21 | The General Hospital Corporation | Polycomb-associated non-coding RNAS |
US9328346B2 (en) | 2010-11-12 | 2016-05-03 | The General Hospital Corporation | Polycomb-associated non-coding RNAs |
US11066673B2 (en) | 2010-11-12 | 2021-07-20 | The General Hospital Corporation | Polycomb-associated non-coding RNAs |
US10358644B2 (en) | 2010-11-12 | 2019-07-23 | The General Hospital Corporation | Polycomb-associated non-coding RNAs |
US9816094B2 (en) | 2010-11-12 | 2017-11-14 | The General Hospital Corporation | Polycomb-associated non-coding RNAs |
US9856479B2 (en) | 2010-11-12 | 2018-01-02 | The General Hospital Corporation | Polycomb-associated non-coding RNAs |
US9920317B2 (en) | 2010-11-12 | 2018-03-20 | The General Hospital Corporation | Polycomb-associated non-coding RNAs |
US9732340B2 (en) | 2011-09-14 | 2017-08-15 | Translate Bio Ma, Inc. | Multimeric oligonucleotides compounds having cleavable linkers |
US9580708B2 (en) | 2011-09-14 | 2017-02-28 | Rana Therapeutics, Inc. | Multimeric oligonucleotides compounds |
US10704046B2 (en) | 2011-09-14 | 2020-07-07 | Translate Bio Ma, Inc. | Multimeric oligonucleotide compounds |
US10093924B2 (en) | 2011-09-14 | 2018-10-09 | Translate Bio Ma, Inc. | Multimetric oligonucleotide compounds |
US9732341B2 (en) | 2011-09-14 | 2017-08-15 | Translate Bio Ma, Inc. | Methods of delivering multiple targeting oligonucleotides to a cell using cleavable linkers |
US10655128B2 (en) | 2012-05-16 | 2020-05-19 | Translate Bio Ma, Inc. | Compositions and methods for modulating MECP2 expression |
US11788089B2 (en) | 2012-05-16 | 2023-10-17 | The General Hospital Corporation | Compositions and methods for modulating MECP2 expression |
US10174323B2 (en) | 2012-05-16 | 2019-01-08 | The General Hospital Corporation | Compositions and methods for modulating ATP2A2 expression |
US10059941B2 (en) | 2012-05-16 | 2018-08-28 | Translate Bio Ma, Inc. | Compositions and methods for modulating SMN gene family expression |
US10058623B2 (en) | 2012-05-16 | 2018-08-28 | Translate Bio Ma, Inc. | Compositions and methods for modulating UTRN expression |
US10837014B2 (en) | 2012-05-16 | 2020-11-17 | Translate Bio Ma, Inc. | Compositions and methods for modulating SMN gene family expression |
US10174315B2 (en) | 2012-05-16 | 2019-01-08 | The General Hospital Corporation | Compositions and methods for modulating hemoglobin gene family expression |
US9790494B2 (en) | 2012-09-14 | 2017-10-17 | Translate Bio Ma, Inc. | Multimeric oligonucleotide compounds having non-nucleotide based cleavable linkers |
US10844375B2 (en) | 2012-09-14 | 2020-11-24 | Translate Bio Ma, Inc. | Multimeric oligonucleotide compounds having non-nucleotide based cleavable linkers |
US10174328B2 (en) | 2013-10-04 | 2019-01-08 | Translate Bio Ma, Inc. | Compositions and methods for treating amyotrophic lateral sclerosis |
US10858650B2 (en) | 2014-10-30 | 2020-12-08 | The General Hospital Corporation | Methods for modulating ATRX-dependent gene repression |
US10758558B2 (en) | 2015-02-13 | 2020-09-01 | Translate Bio Ma, Inc. | Hybrid oligonucleotides and uses thereof |
US10900036B2 (en) | 2015-03-17 | 2021-01-26 | The General Hospital Corporation | RNA interactome of polycomb repressive complex 1 (PRC1) |
Also Published As
Publication number | Publication date |
---|---|
US9090649B2 (en) | 2015-07-28 |
US9719091B2 (en) | 2017-08-01 |
US20160145613A1 (en) | 2016-05-26 |
WO2009146556A1 (en) | 2009-12-10 |
CA2764456C (en) | 2020-09-15 |
ES2643576T3 (en) | 2017-11-23 |
JP2011521652A (en) | 2011-07-28 |
EP2294195B1 (en) | 2017-07-19 |
EP2294195A4 (en) | 2012-12-19 |
EP2294195A1 (en) | 2011-03-16 |
CA2635187A1 (en) | 2009-12-05 |
JP5684116B2 (en) | 2015-03-11 |
CA2764456A1 (en) | 2009-12-10 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US9719091B2 (en) | Oligonucleotide duplexes comprising DNA-like and RNA-like nucleotides and uses thereof | |
TWI727009B (en) | Targeting ligands for therapeutic compounds | |
US20150315591A1 (en) | Nicked or gapped nucleic acid molecules and uses thereof | |
US10144931B2 (en) | Antisense nucleic acids | |
JP2018500027A (en) | Ligand modified double stranded nucleic acid | |
WO2010033225A2 (en) | Compositions and methods for the specific inhibition of gene expression by dsrna possessing modifications | |
US20090069263A1 (en) | 4'-thioarabinonucleotide-containing oligonucleotides, compounds and methods for their preparation and uses thereof | |
JPWO2009102081A1 (en) | Circular single-stranded nucleic acid complex and method for producing the same | |
WO2021122735A1 (en) | Use of sept9 inhibitors for treating hepatitis b virus infection | |
WO2021122869A1 (en) | Use of scamp3 inhibitors for treating hepatitis b virus infection | |
CN114846142A (en) | RNAi agents for inhibiting expression of beta-ENaC, compositions thereof, and methods of use | |
WO2022038211A2 (en) | Use of a1cf inhibitors for treating hepatitis b virus infection | |
US20230193263A1 (en) | Use of sbds inhibitors for treating hepatitis b virus infection | |
CA3218815A1 (en) | Rnai agents for inhibiting expression of mucin 5ac (muc5ac), compositions thereof, and methods of use | |
WO2023150622A2 (en) | Rnai agents for inhibiting expression of coronavirus (cov) viral genomes, compositions thereof, and methods of use | |
WO2021122993A1 (en) | Use of saraf inhibitors for treating hepatitis b virus infection | |
WO2023245060A2 (en) | Rnai agents for inhibiting expression of superoxide dismutase 1 (sod1), compositions thereof, and methods of use | |
WO2021122921A1 (en) | Use of cops3 inhibitors for treating hepatitis b virus infection | |
TW202334416A (en) | Rnai agents for inhibiting expression of matrix metalloproteinase 7 (mmp7), compositions thereof, and methods of use | |
WO2005082921A1 (en) | Method for inhibiting cellular proliferation |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: THE ROYAL INSTITUTION FOR THE ADVANCEMENT OF LEARN Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:DAMHA, MASAD J.;WATTS, JONATHAN K.;DELEAVEY, GLEN;SIGNING DATES FROM 20101126 TO 20101129;REEL/FRAME:025460/0964 |
|
AS | Assignment |
Owner name: LABOPHARM INC., CANADA Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:THE ROYAL INSTITUTION FOR THE ADVANCEMENT OF LEARNING/MCGILL UNIVERSITY;REEL/FRAME:026309/0762 Effective date: 20110511 |
|
AS | Assignment |
Owner name: LABOPHARM INC., CANADA Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:THE ROYAL INSTITUTION FOR THE ADVANCEMENT OF LEARNING/MCGILL UNIVERSITY;REEL/FRAME:026622/0038 Effective date: 20110622 |
|
AS | Assignment |
Owner name: PALADIN LABS INC., CANADA Free format text: MERGER;ASSIGNOR:LABOPHARM INC.;REEL/FRAME:030062/0924 Effective date: 20130101 Owner name: LABOPHARM INC., CANADA Free format text: CHANGE OF NAME;ASSIGNOR:CHIMIGEN INC.;REEL/FRAME:030060/0976 Effective date: 20111116 Owner name: CHIMIGEN INC., CANADA Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:LABOPHARM INC.;REEL/FRAME:030060/0936 Effective date: 20111010 |
|
STCF | Information on status: patent grant |
Free format text: PATENTED CASE |
|
CC | Certificate of correction | ||
MAFP | Maintenance fee payment |
Free format text: PAYMENT OF MAINTENANCE FEE, 4TH YEAR, LARGE ENTITY (ORIGINAL EVENT CODE: M1551); ENTITY STATUS OF PATENT OWNER: LARGE ENTITY Year of fee payment: 4 |
|
AS | Assignment |
Owner name: WILMINGTON TRUST, NATIONAL ASSOCIATION, DELAWARE Free format text: CONFIRMATORY GRANT OF SECURITY INTEREST IN UNITED STATES PATENTS;ASSIGNORS:ENDO GLOBAL VENTURES;ENDO VENTURES LIMITED;PALADIN LABS INC.;REEL/FRAME:053548/0239 Effective date: 20200513 |
|
AS | Assignment |
Owner name: WILMINGTON TRUST, NATIONAL ASSOCIATION, DELAWARE Free format text: CONFIRMATORY GRANT OF SECURITY INTEREST IN UNITED STATES PATENTS;ASSIGNORS:ENDO VENTURES LIMITED;ENDO GLOBAL VENTURES;PALADIN LABS INC.;REEL/FRAME:057538/0893 Effective date: 20200616 |
|
MAFP | Maintenance fee payment |
Free format text: PAYMENT OF MAINTENANCE FEE, 8TH YEAR, LARGE ENTITY (ORIGINAL EVENT CODE: M1552); ENTITY STATUS OF PATENT OWNER: LARGE ENTITY Year of fee payment: 8 |
|
AS | Assignment |
Owner name: PALADIN PHARMA INC., CANADA Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:PALADIN LABS INC.;REEL/FRAME:067203/0281 Effective date: 20240423 |
|
AS | Assignment |
Owner name: PALADIN LABS INC., CANADA Free format text: RELEASE BY SECURED PARTY;ASSIGNOR:WILMINGTON TRUST, NATIONAL ASSOCIATION;REEL/FRAME:067221/0958 Effective date: 20240423 Owner name: ENDO VENTURES LIMITED, IRELAND Free format text: RELEASE BY SECURED PARTY;ASSIGNOR:WILMINGTON TRUST, NATIONAL ASSOCIATION;REEL/FRAME:067221/0958 Effective date: 20240423 Owner name: ENDO VENTURES BERMUDA LIMITED, BERMUDA Free format text: RELEASE BY SECURED PARTY;ASSIGNOR:WILMINGTON TRUST, NATIONAL ASSOCIATION;REEL/FRAME:067221/0958 Effective date: 20240423 Owner name: ENDO GLOBAL VENTURES, BERMUDA Free format text: RELEASE BY SECURED PARTY;ASSIGNOR:WILMINGTON TRUST, NATIONAL ASSOCIATION;REEL/FRAME:067221/0958 Effective date: 20240423 Owner name: ENDO GLOBAL AESTHETICS LIMITED, IRELAND Free format text: RELEASE BY SECURED PARTY;ASSIGNOR:WILMINGTON TRUST, NATIONAL ASSOCIATION;REEL/FRAME:067221/0958 Effective date: 20240423 |
|
AS | Assignment |
Owner name: ENDO OPERATIONS LIMITED, IRELAND Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:PALADIN PHARMA INC.;REEL/FRAME:067245/0714 Effective date: 20240424 |